Anti-ROBO4 antibody

ABSTRACT

The present invention relates to an antibody having an anti-angiogenesis activity. More specifically, the present invention relates to an antibody against ROBO4 and a pharmaceutical composition containing the antibody. An object of the present invention is to provide an anti-ROBO4 antibody having an anti-angiogenesis effect, a pharmaceutical composition or the like comprising the antibody, a method for suppressing angiogenesis using the antibody, etc. Another object of the present invention is to provide a method for producing the antibody. The antibody of the present invention activates the downstream signal of ROBO4 and has a suppressive activity against cell migration induced by VEGF or bFGF. The antibody of the present invention also exhibits an anti-angiogenesis effect in in-vivo models.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/IB2013/053312, filed Apr. 26, 2013.

The Sequence Listing for this application is labeled“Seq-List-replace-2.txt” which was created on Nov. 28, 2016 and is 249KB. The entire content of the sequence listing is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to an antibody having an anti-angiogenesisactivity. More specifically, the present invention relates to anantibody against ROBO4 and a pharmaceutical composition containing theantibody.

BACKGROUND ART

Roundabout homolog 4 (ROBO4), a protein of 110 kDa in molecular weight,has a single-pass transmembrane structure (Non Patent Literature 1) andis known to suppress angiogenesis through the binding to a knownangiogenesis suppressor Slit homolog 2 (Slit2) (Patent Literature 1 andNon Patent Literature 2). Slit2 has been reported to suppress themigration of HUVEC promoted by a vascular endothelial growth factor(VEGF) and has also been reported to suppress the promotion of cellmigration by VEGF or bFGF for ROBO4 gene-transfected vascularendothelial cells (hereinafter, the vascular endothelial cell is alsoreferred to as “EC”) compared with empty vector-transfected EC (PatentLiterature 1 and Non Patent Literatures 2 to 4).

Moreover, it has been reported that the suppressive effect of Slit2 onthe promotion of cell migration, promotion of lumen formation, orincrease in permeability by VEGF is observed in EC derived from awild-type mouse or EC transfected with control siRNA, but not observedin EC derived from a ROBO4 gene-knockout mouse or EC transfected withsiRNA to knock-down the ROBO4 gene (Patent Literature 2 and Non PatentLiteratures 4 to 6). Furthermore, it has been reported that Slit2suppresses, via ROBO4, angiogenesis or increase in vascular permeabilityin mouse models with laser-induced choroidal neovascularization oroxygen-induced retinopathy, which are animal disease models withexudative age-related macular degeneration or diabetic retinopathy(Patent Literature 2 and Non Patent Literature 4).

In spite of these findings, there are also reports showing that ROBO4does not bind to Slit2 (Non Patent Literature 9 and Patent Literature5). It has also been reported as to its functions that ROBO4participates in the promotion of angiogenesis rather than thesuppression of angiogenesis, because the migration or lumen formation ofROBO4 gene-knockout EC is inhibited (Non Patent Literatures 10 and 11).

In clinical practice, ROBO4 has been reported to be highly expressed inintratumoral vessels in liver metastasis from colon cancer,ganglioglioma, bladder cancer, breast cancer, metastatic melanoma,kidney cancer, lung cancer, liver cancer, or colon cancer (PatentLiterature 3 and Non Patent Literatures 1, 3, and 7). Moreover, ROBO4has been reported to be also expressed in blood vessels in thefibrovascular membranes of proliferative diabetic retinopathy patients(Non Patent Literature 8). As such, ROBO4 is expressed in vascularendothelial cells, particularly, endothelial cells in blood vesselsnewly formed in a pathological condition. This may suggest apathological angiogenesis resulting from the high expression of ROBO4,but may also suggest the compensatory expression of ROBO4 forsuppressing pathological angiogenesis.

As described above, ROBO4 is involved in an anti-angiogenesis effect.Thus, an antibody against ROBO4 and a functional fragment thereof arepresumably useful in the treatment of a disease involving angiogenesis.However, it is uncertain whether either an agonistic or antagonisticantibody against ROBO4 suppresses or promotes angiogenesis.

Antibodies described in EP Patent No. 1,565,491 (Patent Literature 4)and WO2008/100805 (Patent Literature 5) are known as the antibodyagainst ROBO4 (hereinafter, referred to as an “anti-ROBO4 antibody”).But none of these antibodies does not show a suppressive or inhibitoryeffect on angiogenesis in vivo.

CITATION LIST Patent Literature

-   [Patent Literature 1] WO2004/003163-   [Patent Literature 2] WO2008/073441-   [Patent Literature 3] WO2002/036771-   [Patent Literature 4] European Patent No. 1,565,491-   [Patent Literature 5] WO2008/100805

Non Patent Literature

-   [Non Patent Literature 1] Genomics, 2002, vol. 79, p. 547-552-   [Non Patent Literature 2] Developmental Biology, 2003, vol. 261, p.    251-267-   [Non Patent Literature 3] Biochemical and Biophysical Research    Communications, 2005, vol. 332, p. 533-541-   [Non Patent Literature 4] Nature Medicine, 2008, No. 14, p. 448-453-   [Non Patent Literature 5] Science Translational Medicine, 2010, vol.    2, p. 23ra19-   [Non Patent Literature 6] Proceedings of the National Academy of    Sciences, 2010, vol. 107, p. 10520-10525-   [Non Patent Literature 7] Oncology Reports, 2006, vol. 15, p.    1437-1443-   [Non Patent Literature 8] Molecular Vision, 2009, vol. 15, p.    1057-1069-   [Non Patent Literature 9] The FASEB Journal, 2005, vol. 19, p.    121-123-   [Non Patent Literature 10] BMC Cell Biology, 2008, vol. 9, p. 61-72-   [Non Patent Literature 11] The FASEB Journal, 2009, vol. 23, p.    513-522

SUMMARY OF INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide an antibody againstROBO4.

A further object of the present invention is to provide a pharmaceuticalcomposition containing an anti-ROBO4 antibody having ananti-angiogenesis effect, etc.

A further object of the present invention is to provide a method forproducing the antibody.

A further object of the present invention is to provide a method forsuppressing angiogenesis using the antibody, etc.

Means for Solving the Problems

The present inventors have conducted diligent studies to attain theobjects and consequently successfully constructed a screening systemthat detects the activation of the downstream signal of ROBO4. Moreover,the present inventors have used the screening system to successfullyobtain a novel anti-ROBO4 monoclonal antibody that activates thedownstream signal of ROBO4, has a suppressive activity against cellmigration induced by various angiogenic factors such as VEGF, bFGF, HGF,PDGF-BB and SDF-1 in ROBO4-expressing EC, and exhibits ananti-angiogenesis effect even in in-vivo models. In this way, thepresent invention has been completed.

Specifically, the present invention relates to:

(1) an antibody having properties described in the following (I) to(III) or a functional fragment thereof:

(I) binding to the ROBO4 protein, preferably with a K_(D) value of1×10⁻⁸ or lower, specifically preferred 5×10⁻⁹ or lower;

(II) suppressing or inhibiting vascular endothelial cell migration inthe absence of a cross-linking antibody in vitro; and

(III) suppressing or inhibiting angiogenesis in vivo;

(2) the antibody or the functional fragment thereof according to (1),wherein the ROBO4 protein is the human ROBO4 protein;

(3) the antibody or the functional fragment thereof according to (1),wherein the ROBO4 protein is a protein consisting of an amino acidsequence of amino acid Nos. 1 to 1007 of SEQ ID NO: 2;

(4) the antibody or the functional fragment thereof according to (1),wherein the ROBO4 protein is a protein consisting of an amino acidsequence of amino acid Nos. 46 to 1007 of SEQ ID NO: 2;

(5) the antibody or the functional fragment thereof according to (3) or(4), wherein the antibody or the functional fragment thereof binds to asite consisting of an amino acid sequence of amino acid Nos. 132 to 209of SEQ ID NO: 2;

(6) the antibody or the functional fragment thereof according to (1),wherein the antibody or the functional fragment thereof is a monoclonalantibody or a functional fragment thereof;

(7) the antibody or the functional fragment thereof according to any oneof (1) to (6), wherein the antibody consists of a heavy chain comprisingCDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 44(FIG. 25), CDRH2 consisting of the amino acid sequence represented bySEQ ID NO: 46 (FIG. 26) or an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 46 by the substitution of one amino acid,and CDRH3 consisting of the amino acid sequence represented by SEQ IDNO: 48 (FIG. 27), and a light chain comprising CDRL1 consisting of theamino acid sequence represented by SEQ ID NO: 50 (FIG. 28) or an aminoacid sequence derived from the amino acid sequence of SEQ ID NO: 50 bythe substitution of one to three amino acid(s), CDRL2 consisting of theamino acid sequence represented by SEQ ID NO: 52 (FIG. 29), and CDRL3consisting of the amino acid sequence represented by SEQ ID NO: 54 (FIG.30);(8) the antibody or the functional fragment thereof according to any oneof (1) to (7), wherein the antibody consists of a heavy chain comprisingCDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 44(FIG. 25), CDRH2 consisting of the amino acid sequence represented bySEQ ID NO: 46 (FIG. 26) or the amino acid sequence represented by SEQ IDNO: 68 (FIG. 44), and CDRH3 consisting of the amino acid sequencerepresented by SEQ ID NO: 48 (FIG. 27), and a light chain comprisingCDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 50(FIG. 28) or the amino acid sequence represented by SEQ ID NO: 70 (FIG.46), CDRL2 consisting of the amino acid sequence represented by SEQ IDNO: 52 (FIG. 29), and CDRL3 consisting of the amino acid sequencerepresented by SEQ ID NO: 54 (FIG. 30);(9) the antibody or the functional fragment thereof according to any oneof (1) to (7), wherein the antibody comprises a heavy chain variableregion consisting of the amino acid sequence represented by SEQ ID NO:31 (FIG. 16), and a light chain variable region consisting of the aminoacid sequence represented by SEQ ID NO: 33 (FIG. 18);(10) the antibody or the functional fragment thereof according to anyone of (1) to (8), wherein the antibody comprises any one heavy chainvariable region selected from the following a) to d) and a light chainvariable region selected from e) and f):

a) a heavy chain (hMAb1-H1-type) variable region consisting of an aminoacid sequence represented by amino acid Nos. 20 to 137 of SEQ ID NO: 56(FIG. 32),

b) a heavy chain (hMAb1-H2-type) variable region consisting of an aminoacid sequence represented by amino acid Nos. 20 to 137 of SEQ ID NO: 58(FIG. 34),

c) a heavy chain (hMAb1-H3-type) variable region consisting of an aminoacid sequence represented by amino acid Nos. 20 to 137 of SEQ ID NO: 60(FIG. 36), and

d) a heavy chain (hMAb1-H4-type) variable region consisting of an aminoacid sequence represented by amino acid Nos. 20 to 137 of SEQ ID NO: 62(FIG. 38); and

e) a light chain (hMAb1-L1-type) variable region consisting of an aminoacid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 64(FIG. 40), and

f) a light chain (hMAb1-L2-type) variable region consisting of an aminoacid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 66(FIG. 42);

(11) the antibody or the functional fragment thereof according to anyone of (1) to (8), wherein the antibody comprises any one of thefollowing combinations 1) to 6) of a heavy chain variable region and alight chain variable region:

1) a heavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 58 (FIG. 34) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40),

2) a heavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 58 (FIG. 34) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 66 (FIG. 42),

3) a heavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 62 (FIG. 38) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 66 (FIG. 42),

4) a heavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 62 (FIG. 38) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40),

5) a heavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 56 (FIG. 32) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40), and

6) a heavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 60 (FIG. 36) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40),

(12) the antibody or the functional fragment thereof according to anyone of (1) to (11), wherein the antibody or the functional fragmentthereof is a chimeric antibody or a functional fragment thereof;

(13) the antibody or the functional fragment thereof according to anyone of (1) to (11), wherein the antibody or the functional fragmentthereof is a humanized antibody or a functional fragment thereof;

(14) the antibody or the functional fragment thereof according to anyone of (1) to (13), wherein the antibody comprises a human IgG1 or humanIgG2 heavy chain constant region;

(15) the antibody or the functional fragment thereof according to anyone of (1) to (6), wherein the antibody or the functional fragmentthereof binds to a site on an antigen recognized by any one antibodyaccording to (7) to (14);

(16) the antibody or the functional fragment thereof according to anyone of (1) to (6), wherein the antibody or the functional fragmentthereof competes with any one antibody according to (7) to (14) for thebinding to ROBO4 protein;

(17) the antibody according to any one of (1) to (8), wherein theantibody comprises any one of the following combinations 1) to 6) of aheavy chain and a light chain:

1) a heavy chain consisting of an amino acid sequence represented byamino acid Nos. 20 to 463 of SEQ ID NO: 58 (FIG. 34) and a light chainconsisting of an amino acid sequence represented by amino acid Nos. 21to 239 of SEQ ID NO: 64 (FIG. 40) (H-1140),

2) a heavy chain consisting of an amino acid sequence represented byamino acid Nos. 20 to 463 of SEQ ID NO: 58 (FIG. 34) and a light chainconsisting of an amino acid sequence represented by amino acid Nos. 21to 239 of SEQ ID NO: 66 (FIG. 42) (H-1143),

3) a heavy chain consisting of an amino acid sequence represented byamino acid Nos. 20 to 463 of SEQ ID NO: 62 (FIG. 38) and a light chainconsisting of an amino acid sequence represented by amino acid Nos. 21to 239 of SEQ ID NO: 66 (FIG. 42) (H-2143),

4) a heavy chain consisting of an amino acid sequence represented byamino acid Nos. 20 to 463 of SEQ ID NO: 62 (FIG. 38) and a light chainconsisting of an amino acid sequence represented by amino acid Nos. 21to 239 of SEQ ID NO: 64 (FIG. 40) (H-2140),

5) a heavy chain consisting of an amino acid sequence represented byamino acid Nos. 20 to 463 of SEQ ID NO: 56 (FIG. 32) and a light chainconsisting of an amino acid sequence represented by amino acid Nos. 21to 239 of SEQ ID NO: 64 (FIG. 40) (H-1040), and

6) a heavy chain consisting of an amino acid sequence represented byamino acid Nos. 20 to 463 of SEQ ID NO: 60 (FIG. 36) and a light chainconsisting of an amino acid sequence represented by amino acid Nos. 21to 239 of SEQ ID NO: 64 (FIG. 40) (H-2040),

(18) an antibody comprising a modified form of the heavy chain of anyone antibody according to (17), wherein said modified form lacks one toseveral carboxyl-terminal amino acid(s) of said heavy chain, preferablyone to eight carboxyl-terminal amino acid(s) of said heavy chain, morepreferably one to two carboxyl-terminal amino acid(s) of said heavychain;(19) the antibody according to any one of (1) to (6), wherein theantibody is 95% or more identical in the amino acid sequence to any oneantibody according to (17); has a K_(D) value of 1×10⁻⁸ or lower for ahuman ROBO4; suppresses or inhibits vascular endothelial cell migrationin the absence of a cross-linking antibody in vitro; and suppresses orinhibits angiogenesis in vivo;(20) the antibody or the functional fragment thereof according to (15),wherein the antibody or the functional fragment thereof is a humanantibody or a functional fragment thereof;(21) a nucleotide sequence selected from the group consisting of thefollowing (I) to (III):(I) a nucleotide sequence comprising a nucleotide sequence encoding thepartial or whole amino acid sequence of the heavy chain or light chainof an antibody according to any one of (1) to (20);(II) a nucleotide sequence consisting of a nucleotide sequencecomprising a nucleotide sequence encoding the partial or whole aminoacid sequence of the heavy chain or light chain of an antibody accordingto any one of (1) to (20); and(III) a nucleotide sequence consisting of a nucleotide sequence encodingthe partial or whole amino acid sequence of the heavy chain or lightchain of an antibody according to any one of (1) to (20);(22) a recombinant vector containing an insert of a nucleotide sequenceaccording to (21);(23) a recombinant cell containing a nucleotide according to (21) or arecombinant vector according to (22) introduced therein;(24) a cell producing an antibody according to any one of (1) to (20),wherein the cell is preferably a mammalian cell, more preferably a CHOcell and even more preferably a CHOK1SV;(25) a method for producing an antibody or a functional fragment thereofaccording to any one of (1) to (20), comprising the following steps (I)and (II):(I) culturing a cell according to (23) or (24); and(II) collecting the antibody or the functional fragment thereofaccording to any one of (1) to (20) from the culture obtained in thestep (I);(26) an antibody or a functional fragment thereof produced by aproduction method according to (25);(27) a modified form of an antibody or a functional fragment thereofaccording to any one of (1) to (20) and (26);(28) a pharmaceutical composition comprising an antibody or a functionalfragment thereof according to any one of (1) to (20) and (26) or amodified form according to (27) as an active ingredient;(29) the pharmaceutical composition according to (28), wherein thepharmaceutical composition is an agent for treating or preventing anangiogenic disease;(30) the pharmaceutical composition according to (28), wherein theangiogenic disease is exudative age-related macular degeneration,diabetic retinopathy, macular edema, benign or malignant tumor,atherosclerosis, retrolental fibroplasia, angioma, chronic inflammation,ocular neovascular disease, proliferative retinopathy, neovascularglaucoma, immune rejection of a corneal tissue transplant or othertissue transplants, rheumatoid arthritis, psoriasis, acute inflammation,sepsis, or obesity;(31) the pharmaceutical composition according to (28), wherein theangiogenic disease is exudative age-related macular degeneration,diabetic retinopathy, macular edema, retrolental fibroplasia, ocularneovascular disease, proliferative retinopathy, neovascular glaucoma, orimmune rejection of a corneal tissue transplant;(32) an angiogenesis inhibitor comprising an antibody or a functionalfragment thereof according to any one of (1) to (20) and (26) or amodified form according to (27) as an active ingredient;(33) a method for treating or preventing an angiogenic diseasecomprising administering to a subject in need thereof an effectiveamount of an antibody or a functional fragment thereof according to anyone of (1) to (20) and (26) or a modified form according to (27), or thecomposition according to any one of (28) to (31), preferably wherein theangiogenic disease is the angiogenic disease in an individual having theexpressed ROBO4 protein; and(34) the pharmaceutical composition according to any one of claims 28 to31, wherein said composition is used in combination with a furthertherapeutic or prophylactic agent, preferably wherein said agent is ananti-angiogenesis drug, anti-inflammatory drug, and/or an anticancerdrug.

Advantageous Effects of Invention

According to the present invention, a therapeutic agent or the like foran angiogenic disease containing an antibody that binds to ROBO4 and hasan anti-angiogenesis effect can be obtained.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the presence or absence of change inreporter activity against NF-κB, GAS, ISRE, IL-8 promoter, or TCF asresponse elements in the transient expression of human ROBO4 in HEK293cells. The error bar in the diagram represents standard deviation (n=3).

FIG. 2 is a diagram showing the presence or absence of change in IL-8promoter activity in the transient expression of full-length human ROBO4or an intracellular region deletion variant of human ROBO4 in HEK293cells. The error bar in the diagram represents standard deviation (n=5or 10).

FIG. 3 is a diagram showing change in IL-8 promoter activity in humanROBO4-transfected HEK293 cells caused by an anti-ROBO4 antibody MAb1.The error bar in the diagram represents standard deviation (n=3).

FIG. 4 is a diagram showing change in IL-8 promoter activity in humanROBO4-transfected HEK293 cells caused by an anti-ROBO4 antibody MAb2,MAb3, or MAb4. The error bar in the diagram represents standarddeviation (n=3).

FIG. 5 is a diagram showing change in the migratory capacity of HUVEC inthe presence of VEGF or bFGF caused by the anti-ROBO4 antibody MAb1. Theerror bar in the diagram represents standard deviation (n=3 or 4).

FIG. 6 is a diagram showing change in the migratory capacity of HUVEC inthe presence of bFGF caused by the anti-ROBO4 antibody MAb2, MAb3, orMAb4. The error bar in the diagram represents standard deviation (n=4).

FIG. 7 is a diagram showing the presence or absence of the bindingactivity of the anti-ROBO4 antibody MAb1 against human ROBO4, mouseROBO4, rat ROBO4, or cynomolgus monkey ROBO4.

FIG. 8 is a diagram showing the presence or absence of the bindingactivity of the anti-ROBO4 antibody MAb1 against human ROBO1, humanROBO2, or human ROBO3. The upper boxes show the results about MAb1, andthe lower boxes show the results about a positive control antibody withwhich the expression of these proteins on cell surface was confirmed.

FIG. 9 is a diagram showing the presence or absence of the bindingactivity of the anti-ROBO4 antibody MAb1 against an extracellularregion/domain deletion variant of human ROBO4. The upper boxes show theresults about MAb1, and the lower boxes show the results about ananti-FLAG antibody with which the expression of these proteins on cellsurface was confirmed.

FIG. 10 is a diagram showing change in angiogenesis in laser-inducedchoroidal neovascularization models caused by the anti-ROBO4 antibodyMAb1. The error bar in the diagram represents standard deviation (n=4eyes), and ▪ or □ shows the results of each eye.

FIG. 11 is a diagram showing change in IL-8 promoter activity in humanROBO4-transfected HEK293 cells caused by an anti-ROBO4 chimeric antibodycMAb1-1 or cMAb1-2. The error bar in the diagram represents standarddeviation (n=3).

FIG. 12 is a diagram showing change in the migratory capacity of HUVECin the presence of bFGF caused by the anti-ROBO4 chimeric antibodycMAb1-1 or cMAb1-2. The error bar in the diagram represents standarddeviation (n=4).

FIG. 13 shows cDNA encoding full-length human ROBO4 (SEQ ID NO: 1).

FIG. 14 shows the full-length amino acid sequence of human ROBO4 (SEQ IDNO: 2).

FIG. 15 shows the nucleotide sequence of cDNA encoding the heavy chainvariable region of MAb1 (SEQ ID NO: 30).

FIG. 16 shows the amino acid sequence of the MAb1 heavy chain variableregion (SEQ ID NO: 31).

FIG. 17 shows the nucleotide sequence of cDNA encoding the light chainvariable region of MAb1 (SEQ ID NO: 32).

FIG. 18 shows the amino acid sequence of the MAb1 light chain variableregion (SEQ ID NO: 33).

FIG. 19 shows the nucleotide sequence of cDNA encoding the light chainof cMAb1 (SEQ ID NO: 37).

FIG. 20 shows the amino acid sequence of the cMAb1 light chain (SEQ IDNO: 38).

FIG. 21 shows the nucleotide sequence of cDNA encoding the heavy chainof cMAb1-1 (SEQ ID NO: 39).

FIG. 22 shows the amino acid sequence of the cMAb1-1 heavy chain (SEQ IDNO: 40).

FIG. 23 shows the nucleotide sequence of cDNA encoding the heavy chainof cMAb1-2 (SEQ ID NO: 41).

FIG. 24 shows the amino acid sequence of the cMAb1-2 heavy chain (SEQ IDNO: 42).

FIG. 25 shows the amino acid sequence of the heavy chain CDRH1 of MAb1(SEQ ID NO: 44).

FIG. 26 shows the amino acid sequence of the heavy chain CDRH2 of MAb1(SEQ ID NO: 46).

FIG. 27 shows the amino acid sequence of the heavy chain CDRH3 of MAb1(SEQ ID NO: 48).

FIG. 28 shows the amino acid sequence of the light chain CDRL1 of MAb1(SEQ ID NO: 50).

FIG. 29 shows the amino acid sequence of the light chain CDRL2 of MAb1(SEQ ID NO: 52).

FIG. 30 shows the amino acid sequence of the light chain CDRL3 of MAb1(SEQ ID NO: 54).

FIG. 31 shows the nucleotide sequence of cDNA encoding a hMAb1-H1-typeheavy chain (SEQ ID NO: 55).

FIG. 32 shows the amino acid sequence of the hMAb1-H1-type heavy chain(SEQ ID NO: 56).

FIG. 33 shows the nucleotide sequence of cDNA encoding a hMAb1-H2-typeheavy chain (SEQ ID NO: 57).

FIG. 34 shows the amino acid sequence of the hMAb1-H2-type heavy chain(SEQ ID NO: 58).

FIG. 35 shows the nucleotide sequence of cDNA encoding a hMAb1-H3-typeheavy chain (SEQ ID NO: 59).

FIG. 36 shows the amino acid sequence of the hMAb1-H3-type heavy chain(SEQ ID NO: 60).

FIG. 37 shows the nucleotide sequence of cDNA encoding a hMAb1-H4-typeheavy chain (SEQ ID NO: 61).

FIG. 38 shows the amino acid sequence of the hMAb1-H4-type heavy chain(SEQ ID NO: 62).

FIG. 39 shows the nucleotide sequence of cDNA encoding a hMAb1-L1-typelight chain (SEQ ID NO: 63).

FIG. 40 shows the amino acid sequence of the hMAb1-L1-type light chain(SEQ ID NO: 64).

FIG. 41 shows the nucleotide sequence of cDNA encoding a hMAb1-L2-typelight chain (SEQ ID NO: 65).

FIG. 42 shows the amino acid sequence of the hMAb1-L2-type light chain(SEQ ID NO: 66).

FIG. 43 shows the amino acid sequence of CDRH1 of the hMAb1-H2- orhMAb1-H4-type heavy chain (SEQ ID NO: 67).

FIG. 44 shows the amino acid sequence of CDRH2 of the hMAb1-H2- orhMAb1-H4-type heavy chain (SEQ ID NO: 68).

FIG. 45 shows the amino acid sequence of CDRH3 of the hMAb1-H2- orhMAb1-H4-type heavy chain (SEQ ID NO: 69).

FIG. 46 shows the amino acid sequence of CDRL1 of the hMAb1-L2-typelight chain (SEQ ID NO: 70).

FIG. 47 shows the amino acid sequence of CDRL2 of the hMAb1-L2-typelight chain (SEQ ID NO: 71).

FIG. 48 shows the amino acid sequence of CDRL3 of the hMAb1-L2-typelight chain (SEQ ID NO: 72).

FIG. 49 is a diagram showing change in IL-8 promoter activity in humanROBO4-transfected HEK293 cells caused by H-1040, H-1143, H-1140, H-2040,H-2143, or H-2140.

FIG. 50 is a diagram showing change in the migratory capacity of HUVECin the presence of bFGF caused by H-1143, H-2140, or H-2143. The errorbar in the diagram represents standard deviation (n=4).

FIG. 51 is a diagram showing the cross-species reactivity of H-1143.

FIG. 52 is a diagram showing the cross-species reactivity of H-2140.

FIG. 53 is a diagram showing the cross-species reactivity of H-2143.

FIG. 54 is a diagram showing the binding specificity of H-1143, H-2140,or H-2143.

FIG. 55 is a diagram showing change in angiogenesis in laser-inducedchoroidal neovascularization models caused by H-2143. The error bar inthe diagram represents standard deviation (n=3-4 eyes).

MODE FOR CARRYING OUT THE INVENTION 1. Definition

In the present invention, “gene” means nucleotide(s) or nucleotidesequence comprising a nucleotide sequence encoding the amino acids of aprotein, or its complementary strand. The “gene” is meant to include,for example, a polynucleotide, an oligonucleotide, DNA, mRNA, cDNA, andcRNA as the nucleotide sequence comprising a nucleotide sequenceencoding the amino acids of a protein, or its complementary strand. Sucha gene is a single-stranded, double-stranded, or triple or more strandednucleotide sequence, and the “gene” is also meant to include anassociate of DNA and RNA strands, a mixture of ribonucleotides (RNAs)and deoxyribonucleotides (DNAs) on one nucleotide strand, and adouble-stranded or triple or more stranded nucleotide sequencecomprising such a nucleotide strand. Examples of the “ROBO4 gene” of thepresent invention can include DNA, mRNA, cDNA, and cRNA comprising anucleotide sequence encoding the amino acid sequence of the ROBO4protein.

In the present invention, the term “nucleotide(s)” or “nucleotidesequence” has the same meaning as in a “nucleic acid” and is also meantto include, for example, DNA, RNA, a probe, an oligonucleotide, apolynucleotide, and a primer. Such a nucleotide sequence is asingle-stranded, double-stranded or triple or more stranded nucleotide,and the “nucleotide” sequence is also meant to include an associate ofDNA and RNA strands, a mixture of ribonucleotides (RNAs) anddeoxyribonucleotides (DNAs) on one nucleotide strand, and an associateof two strands or three or more strands comprising such a nucleotidestrand.

In the present invention, the terms “polypeptide”, “peptide”, and“protein” have the same meaning.

In the present invention, the “antigen” is also used as the meaning ofan “immunogen”.

In the present invention, the “cell” also includes various cells derivedfrom animal individuals, subcultured cells, primary cultured cells, celllines, recombinant cells, and the like.

In the present invention, an antibody recognizing the ROBO4 protein isalso referred to as an “anti-ROBO4 antibody”. The “anti-ROBO4 antibody”includes an anti-ROBO4 chimeric antibody, an anti-ROBO4 humanizedantibody, an anti-ROBO4 human antibody, and the like.

In the present invention, the “functional fragment of the antibody”means an antibody fragment that exerts at least one of the functions,e.g., the binding affinity (K_(D) value) of the original antibody.Examples of the “functional fragment of the antibody” can include, butnot limited to, Fab, F(ab′)2, scFv, Fab′, single-chain immunoglobulin,and the like. Such a functional fragment of the antibody may be obtainedby the treatment of a full-length molecule of the antibody protein withan enzyme such as papain or pepsin or may be a recombinant proteinproduced in an appropriate host cell using a recombinant gene. Preferred“functional fragments” also have at least one of the biologicalactivities of the original antibody.

Further, in the context of the present invention, a nucleotide sequenceencoding “the partial amino acid sequence” of the heavy or light chainis or includes a nucleotide sequence encoding a “functional fragment ofthe antibody” as defined herein above.

In the present invention, the “site” to which an antibody binds, i.e.,the “site” recognized by an antibody, means a partial peptide or partialconformation on an antigen bound or recognized by the antibody. In thepresent invention, such a site is also called an epitope or anantibody-binding site. Examples of the site on the ROBO4 protein boundor recognized by the anti-ROBO4 antibody of the present invention caninclude a partial peptide or partial conformation on the ROBO4 protein.

The heavy and light chains of an antibody molecule are known to eachhave three complementarity determining regions (CDRs). Thecomplementarity determining regions are also called hypervariabledomains. They are located in the variable regions of the antibody heavyand light chains. These sites have a particularly highly variableprimary structure and are usually separated at three positions on therespective primary structures of heavy and light chain polypeptidestrands. In the present invention, the complementarity determiningregions of the antibody are referred to as CDRH1, CDRH2, and CDRH3 fromthe amino terminus of the heavy chain amino acid sequence as to thecomplementarity determining regions of the heavy chain and as CDRL1,CDRL2, and CDRL3 from the amino terminus of the light chain amino acidsequence as to the complementarity determining regions of the lightchain. These sites are proximal to each other on the three-dimensionalstructure and determine specificity for the antigen to be bound.

In the present invention, the “antibody mutant” means a polypeptide thathas an amino acid sequence derived from the amino acid sequence of theoriginal antibody by the substitution, deletion, addition, and/orinsertion (hereinafter, collectively referred to as “mutation”) of aminoacid(s) and binds to the ROBO4 protein of the present invention. Thenumber of the mutated amino acids in such an antibody mutant is 1 to 2,1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to12, 1 to 15, 1 to 20, 1 to 25, 1 to 30, 1 to 40, or 1 to 50. Such anantibody mutant is also encompassed by the “antibody” of the presentinvention.

In the present invention, the term “several” in “1 to several” refers to2 to 10, preferably 2 to 8, more preferably 2.

Examples of activities or properties exerted by the antibody of thepresent invention can include biological activities or physicochemicalproperties and can specifically include various biological activities, abinding activity against an antigen or an epitope, stability duringproduction or storage, and thermal stability.

In the present invention, the phrase “hybridizing under stringentconditions” means hybridization under conditions involving hybridizationat 65° C. in a solution containing 5×SSC, followed by washing at 65° C.for 20 minutes in an aqueous solution containing 2×SSC-0.1% SDS, at 65°C. for 20 minutes in an aqueous solution containing 0.5×SSC-0.1% SDS,and at 65° C. for 20 minutes in an aqueous solution containing0.2×SSC-0.1% SDS, or hybridization under conditions equivalent thereto.SSC means an aqueous solution of 150 mM NaCl-15 mM sodium citrate, andn×SSC means SSC with an n-fold concentration.

2. ROBO4 Protein

In the present specification, the terms “ROBO4” and “ROBO4 protein” areused as the same meaning.

(2-1) Property

The ROBO4 protein of the present invention has the following properties:

(i) ROBO4 has a molecular weight of approximately 110 kDa and asingle-pass transmembrane structure and is a receptor protein of theSLIT2 protein involved in angiogenesis. Any ROBO4 protein of the presentinvention can be found in a form liberated from a membrane such as acell membrane and may be in a form bound to a membrane such as a cellmembrane. In this context, the molecular weight means an apparentmolecular weight under the non-reducing conditions of SDS-PAGE. TheN-terminal extracellular region of ROBO4 contains twoimmunoglobulin-like domains (hereinafter, referred to as “Ig-likedomains”) and two fibronectin type III domains, while its C-terminalintracellular region contains a protein-rich region. In the presentspecification, these two immunoglobulin-like domains are referred to asIg-like domain 1 and Ig-like domain 2, respectively, from the aminoterminus. The human ROBO4 protein consists of an amino acid sequence ofamino acid Nos. 28 to 1007 of SEQ ID NO: 2. Amino acid Nos. 1 to 27 ofSEQ ID NO: 2 represent a secretory signal; amino acid Nos. 28 to 467thereof represent an extracellular region; amino acid Nos. 46 to 131thereof represent Ig-like domain 1; amino acid Nos. 137 to 224 thereofrepresent Ig-like domain 2; amino acid Nos. 252 to 340 thereof representfibronectin type III domain 1; amino acid Nos. 347 to 438 thereofrepresent fibronectin type III domain 2; amino acid Nos. 468 to 490thereof represent a region in the cell membrane; and amino acid Nos. 491to 1007 thereof represent an intracellular region.(ii) ROBO4 has an anti-angiogenesis effect. In the present invention,the term “anti-angiogenesis” means that the molecule directly orindirectly suppresses and/or inhibits angiogenesis by itself, incollaboration with another factor, or as an associate with anotherfactor. The anti-angiogenesis effect can be evaluated, for example, witha suppressive effect on increase in vascular permeability, cellmigration promoting activity, or lumen formation activity by VEGF as anindex.(iii) ROBO4 comprises an amino acid sequence described in any one of thefollowing (a) to (e) (hereinafter, referred to as a “ROBO4 amino acidsequence”), consists of an amino acid sequence comprising the ROBO4amino acid sequence, or consists of the ROBO4 amino acid sequence:(a) the amino acid sequence represented by SEQ ID NO: 2 (FIG. 14);(b) the amino acid sequence of a polypeptide that exhibits 80% or more,82% or more, 84% or more, 86% or more, 88% or more, 90% or more, 92% ormore, 94% or more, 96% or more, 98% or more, or 99% or more sequenceidentity to the amino acid sequence represented by SEQ ID NO: 2 (FIG.14) and exhibits an anti-angiogenesis effect;(c) the amino acid sequence of a polypeptide that comprises an aminoacid sequence represented by SEQ ID NO: 2 (FIG. 14) having asubstitution, deletion, addition, or insertion of 1 to 50, 1 to 45, 1 to40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid(s) and suppressesangiogenesis;(d) the amino acid sequence of a polypeptide that comprises an aminoacid sequence of SEQ ID NO: 2 having the deletion of amino acid Nos. 1to 45 or 1 to 131 and suppresses angiogenesis; and(e) the amino acid sequence of a polypeptide that is encoded by thenucleotide sequence of a nucleotide hybridizing under stringentconditions to a nucleotide having a nucleotide sequence complementary toa nucleotide sequence encoding the amino acid sequence represented bySEQ ID NO: 2 (FIG. 14) and suppresses angiogenesis.

The ROBO4 protein may be present as the whole or a portion of a homo orhetero oligo associate constituted of two or more subunits.

The amino acid sequence and/or other properties of the ROBO4 protein maybe neither the same nor homogeneous in an individual, a tissue, a bodyfluid, a cell, a ROBO4 protein-containing fraction, a purified orpartially purified ROBO4 protein preparation, or the like, or among aplurality of individuals, tissues, cells, ROBO4 protein-containingfractions, or ROBO4 protein preparations. One individual, tissue, bodyfluid, cell, ROBO4 protein-containing fraction, purified or partiallypurified ROBO4 protein preparation, or the like may contain plural typesof ROBO4 proteins differing in amino acid sequence and/or property.Alternatively, a plurality of individuals, tissues, cells, ROBO4protein-containing fractions, or ROBO4 protein preparations may differin the amino acid sequence and/or other properties of the ROBO4 protein.Even such proteins differing in amino acid sequence and/or propertiesfrom each other are all encompassed by the “ROBO4 protein” of thepresent invention as long as they possess the properties described abovein (i) to (iii).

(iv) The ROBO4 protein of the present invention can be obtained from thetissue of a vertebrate, preferably a mammal, more preferably a rodentsuch as a mouse or a rat or a human, even more preferably tissues of ahuman or a mouse, cells derived from such a tissue, cultures of suchcells, and the like. Such a tissue and cells are not particularlylimited as long as they contain the ROBO4 protein. Examples thereof caninclude joint tissues, blood, lymph, thymus glands, spleens, and cellsderived from any of them. Preferable tissues and cells are tissues andcells derived from animals or patients having angiogenesis. However, theorigin of the ROBO4 protein of the present invention is not limited tothose described above, and the ROBO4 protein of the present invention isalso meant to include even ROBO4 proteins derived from other animalspecies, other tissues, other cells, or the like as long as they possessthe properties described above in (i) to (iii).

The ROBO4 protein of the present invention may be any of native andrecombinant proteins. The ROBO4 protein is also meant to include fusionproducts with another peptide or protein such as a carrier or a tag. TheROBO4 protein is further meant to include forms provided with chemicalmodification including the addition of a polymer such as PEG and/or withbiological modification including sugar chain modification. Moreover,the ROBO4 protein of the present invention is meant to include a ROBO4protein fragment. A ROBO4 protein fragment possessing the propertydescribed above in (ii) is called a functional fragment of the ROBO4protein.

(2-2) ROBO4 Gene

The ROBO4 gene of the present invention comprises a nucleotide sequencedescribed in any one of the following (a) to (c) (hereinafter, referredto as a “ROBO4 gene sequence”), consists of a nucleotide sequencecomprising the ROBO4 gene sequence, or consists of the ROBO4 genesequence:

(a) the nucleotide sequence represented by SEQ ID NO: 1 (FIG. 13);

(b) a nucleotide sequence that hybridizes under stringent conditions toa nucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence represented by SEQ ID NO: 1 (FIG. 13) and encodesthe amino acid sequence of a polypeptide suppressing angiogenesis; and(c) a nucleotide sequence that comprises a nucleotide sequencerepresented by SEQ ID NO: 1 (FIG. 13) having a substitution, deletion,addition, or insertion of 1 to 150, 1 to 140, 1 to 130, 1 to 120, 1 to110, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to5, 1 to 4, 1 to 3, 1 or 2, or 1 base(s) and encodes the amino acidsequence of a polypeptide suppressing angiogenesis.

The ROBO4 gene is overexpressed in blood vessels in fibrovascularmembranes or intratumoral vessels of patients with a disease accompaniedby angiogenesis, for example, proliferative diabetic retinopathy. Inaddition, the ROBO4 gene seems to be overexpressed in tissue or bloodfractions derived from patients affected with a disease considered toinvolve angiogenesis, such as exudative age-related maculardegeneration, macular edema, atherosclerosis, retrolental fibroplasia,angioma, chronic inflammation, ocular neovascular disease, proliferativeretinopathy, neovascular glaucoma, immune rejection of a corneal tissuetransplant or other tissue transplants, rheumatoid arthritis, psoriasis,acute inflammation, sepsis, or obesity, or from model animals of thesediseases.

The expression and expression level of the ROBO4 gene may be assayedwith any of a ROBO4 gene transcription product and the ROBO4 protein asan index and can be determined by RT-PCR, Northern blot hybridization,or the like for the upper index and by immunoassay (e.g., enzyme-linkedimmuno-sorbent assay; hereinafter, referred to as “ELISA”) or the likefor the latter index.

(2-3) Preparation of Protein

The ROBO4 protein of the present invention can be purified or isolatedfrom animal tissues (including body fluids), cells derived from thetissues, or cultures of the cells and prepared by gene recombination,in-vitro translation, chemical synthesis, etc.

(2-3-1) Purification or Isolation of Native ROBO4

The native ROBO4 protein can be purified or isolated from, for example,tissues (including body fluids, cells, etc.) derived from patients ornon-human animals affected with an angiogenic disease such as exudativeage-related macular degeneration, diabetic retinopathy, macular edema,benign or malignant tumor, atherosclerosis, retrolental fibroplasia,angioma, chronic inflammation, ocular neovascular disease, proliferativeretinopathy, neovascular glaucoma, immune rejection of a corneal tissuetransplant or other tissue transplants, rheumatoid arthritis, psoriasis,acute inflammation, sepsis, or obesity, cells derived from the tissues,or cultures of the cells. Such non-human animals also include modelanimals of these diseases. Animals subjected to model preparation arenot particularly limited as long as they are vertebrates. The animalsare preferably mammals, more preferably rodents such as mice or rats,even more preferably mice or rats. The tissues and cells of suchpatients or model animals are not particularly limited as long as theycontain the ROBO4 protein. Examples thereof can include joint tissues,blood, lymph, thymus glands, spleens, and cells derived from any ofthem. Preferable tissues and cells are derived from patients or modelanimals having angiogenesis or exhibiting similar symptoms. However, theorigin of the ROBO4 protein of the present invention is not limited tothose described above, and the ROBO4 protein of the present inventionmay be derived from other animal species, other tissues, other cells, orthe like.

Purification or isolation from such tissues, cells, cell cultures, orthe like can be performed by the combination of approaches well known bythose skilled in the art, such as fractionation and chromatography. Suchapproaches include, but not limited to, salting out, gel filtration,ion-exchange chromatography, affinity chromatography, hydrophobicchromatography, normal-phase or reverse-phase chromatography, and thelike. An affinity gel cross-linked with an anti-ROBO4 monoclonalantibody can be prepared and loaded to a column to thereby prepare acolumn for affinity chromatography. A crude or partially purifiedfraction containing the ROBO4 protein is added to such a column.Subsequently, non-specific adsorbed matter is removed with sterilizedphosphate-buffered saline (PBS), and a buffer solution for elution canthen be added thereto to thereby selectively collect the ROBO4 protein.The solution containing the ROBO4 protein can be subjected to gelfiltration or to buffer replacement and/or concentration using aconcentrator such as Centriprep.

(2-3-2) Preparation of Recombinant ROBO4 Protein

The ROBO4 protein of the present invention can also be prepared in arecombinant form. Specifically, host cells are transfected with a geneencoding the amino acid sequence of the ROBO4 protein or a ROBO4 proteinfragment, and the ROBO4 protein can be collected from cultures of thecells. For example, the ROBO4 gene or its fragment is inserted into anexpression vector. Subsequently, prokaryotic or eukaryotic host cellsare transfected with the resulting recombinant vector, and the obtainedrecombinant cells can be incubated to thereby express the ROBO4 protein.An expression pattern known in the art, such as secretion expression,intracellular expression of soluble forms, or an inclusion body methodcan be used. Also, the ROBO4 protein can be expressed not only as amolecule having the same amino terminus (N-terminus) and/or carboxyterminus (C-terminus) as native ones, but also as a fusion protein witha secretory signal, an intracellular localization signal, a tag foraffinity purification, or a partner peptide. The ROBO4 protein can bepurified or isolated from such recombinant cell cultures by theappropriate combination of operations such as fractionation andchromatography described in (2-3-1) Purification or isolation of nativeROBO4 protein.

The ROBO4 gene or its fragment can be prepared by a method well known bythose skilled in the art.

Examples thereof can include: polymerase chain reaction (hereinafter,referred to as “PCR”; Saiki, R. K., et al., Science (1988) 239, p.487-489) with a ROBO4 cDNA expression library as a template using oneset of primers capable of specifically amplifying the sequence; reversetranscription PCR (hereinafter, referred to as “RT-PCR”) with an mRNAfraction for ROBO4 expression as a template using a primer capable ofreverse-transcribing the sequence and one set of primers capable ofspecifically amplifying the sequence; expression cloning usingimmunoassay; and cDNA cloning using the partial amino acid sequence of apurified ROBO4 protein.

(2-3-3) In-Vitro Translation

The ROBO4 protein of the present invention can also be prepared byin-vitro translation. Such a translation method is not particularlylimited as long as it is a method using a cell-free translation systeminvolving enzymes necessary for transcription and translation,substrates, and energy substances. Examples thereof can include a methodusing Rapid Translation System (RTS) manufactured by Roche Diagnostics.

(2-3-4) Chemical Synthesis

The ROBO4 protein of the present invention can also be prepared bychemical synthesis. Examples of the chemical synthesis method caninclude solid-phase peptide synthesis methods such as Fmoc synthesis andBoc synthesis methods.

3. Anti-ROBO4 Antibody

(3-1) Type of Anti-ROBO4 Antibody

The antibody of the present invention may be any of monoclonal andpolyclonal antibodies. Examples of the monoclonal antibody of thepresent invention can include a non-human animal-derived antibody(non-human animal antibody), a human-derived antibody (human antibody),a chimeric antibody, and a humanized antibody, preferably a chimericantibody, a humanized antibody, and a human-derived antibody (humanantibody), more preferably a humanized antibody, and a human-derivedantibody (human antibody).

Examples of the non-human animal antibody can include antibodies derivedfrom vertebrates such as mammals and birds. Examples of themammal-derived antibody can include rodent-derived antibodies such asmouse antibodies and rat antibodies. Examples of the bird-derivedantibody can include chicken antibodies.

Examples of the chimeric antibody can include, but not limited to, anantibody comprising non-human animal antibody-derived variable regionsbound with human antibody (human immunoglobulin) constant regions.Examples of the non-human animal antibody-derived variable regions caninclude heavy and light chain variable regions derived from MAb1described later.

Examples of the humanized antibody can include, but are not limited to,a human antibody (human immunoglobulin variable regions) grafted withCDRs in the variable regions of a non-human animal antibody, a humanantibody grafted with the CDRs as well as with partial sequences offramework regions of a non-human animal antibody, and an antibody havinghuman antibody amino acid(s) replaced for one or two or more non-humananimal antibody-derived amino acid(s) in any of these humanizedantibodies. Examples of the CDRs in the variable regions of a non-humananimal antibody can include CDRH1 to 3 in the heavy chain variableregion and CDRL1 to 3 in the light chain variable region derived fromMAb1 described later.

The human antibody is not particularly limited as long as it is anantibody recognizing the antigen of the present invention. Examplesthereof can include a human antibody binding to the same site as thatbound by an antibody having the antibody CDRs of the present invention,and a human antibody binding to the same site on ROBO4 as that bound byMAb1 described above.

The antibody according to the present invention may be an antibodyconstituted of sites derived from a plurality of different antibodies.Examples thereof can include an antibody comprising heavy and/or lightchains exchanged among a plurality of different antibodies, an antibodycomprising full-length heavy and/or light chains exchanged thereamong,an antibody comprising variable or constant regions exchangedthereamong, and an antibody comprising all or some CDRs exchangedthereamong. The heavy and light chain variable regions of the chimericantibody may be derived from different antibodies of the presentinvention. CDRH1 to 3 and CDRL1 to 3 in the heavy and light chainvariable regions of the humanized antibody may be derived from two ormore different antibodies of the present invention. CDRH1 to 3 and CDRL1to 3 in the heavy and light chain variable regions of the human antibodymay be the combination of CDRs carried by two or more differentantibodies of the present invention.

The isotype of the monoclonal antibody of the present invention is notparticularly limited, and examples thereof can include IgG such as IgG1,IgG2, IgG3, and IgG4, IgM, IgA such as IgA1 and IgA2, IgD, and IgE andcan preferably include IgG and IgM, more preferably IgG2. The isotypeand subclass of the monoclonal antibody can be determined by, forexample, an Ouchterlony test, ELISA, radio immunoassay (hereinafter,referred to as “RIA”). A commercially available kit for identification(Mouse Typer Kit manufactured by Bio-Rad Laboratories, Inc., RATMONOCLONAL ANTIBODY ISOTYPING TEST KIT manufactured by AbD Serotec,etc.) may be used.

(3-2) Binding Specificity of Anti-ROBO4 Antibody

The antibody of the present invention recognizes the ROBO4 protein. Inother words, the antibody of the present invention binds to the ROBO4protein. Such an antibody is referred to as an “anti-ROBO4 antibody”.Moreover, the preferable antibody of the present invention specificallyrecognizes the ROBO4 protein. In other words, the preferable antibody ofthe present invention specifically binds to the ROBO4 protein.Furthermore, the more preferable antibody of the present inventionspecifically binds to an Ig-like domain carried by the ROBO4 protein.Examples of such an Ig-like domain can include Ig-like domain 1 andIg-like domain 2. The more preferable antibody of the present inventionrecognizes a region consisting of an amino acid sequence of amino acidNos. 132 to 209 of SEQ ID NO: 2. The antibody of the present inventionbinds to a human ROBO4 protein, a monkey, preferably cynomolgus monkeyROBO4 protein, and a rabbit ROBO4 protein, but does not bind to mouseand rat ROBO4 proteins (Cross-species reactivity in Example 4)-3 andExample 11)-4).

In the present invention, the “specific recognition”, i.e., “specificbinding”, means binding which is not non-specific adsorption. Examplesof criteria for determination on whether binding is specific or not caninclude a dissociation constant (hereinafter, referred to as “K_(D)”).The preferable antibody of the present invention has a K_(D) value of1×10⁻⁵ or lower, 5×10⁻⁶ or lower, 2×10⁻⁶ or lower, or 1×10⁻⁶ or lower,more preferably 5×10⁻⁷ or lower, 2×10⁻⁷ or lower, or 1×10⁻⁷ or lower,even more preferably 5×10⁻⁸ or lower, 2×10⁻⁸ or lower, or 1×10⁻⁸ orlower, further more preferably 5×10⁻⁹ or lower, 2×10⁻⁹ or lower, or1×10⁻⁹ or lower, most preferably 5×10⁻¹⁰ or lower, 2×10⁻¹⁰ or lower, or1×10⁻¹⁰ or lower for the ROBO4 protein. More specifically, thepreferable antibody of the present invention has a K_(D) value of 2×10⁻⁸or lower, more preferably 1×10⁻⁸ or lower, even more preferably 5×10⁻⁹or lower for the ROBO4 protein.

In the present invention, the binding of the antibody to the antigen canbe assayed or determined by ELISA, RIA, surface plasmon resonance(hereinafter, referred to as “SPR”) analysis, or the like. Examples ofequipment used in the SPR analysis can include BIAcore™ (manufactured byGE Healthcare Bio-Sciences Corp.), ProteOn™ (manufactured by Bio-RadLaboratories, Inc.), SPR-Navi™ (manufactured by BioNavis Oy Ltd.),Spreeta™ (manufactured by Texas Instruments Inc.), SPRi-PlexII™(manufactured by Horiba, Ltd.), and Autolab SPR™ (manufactured byMetrohm). The binding of the antibody to the antigen expressed on cellsurface can be assayed by flow cytometry or the like.

(3-3) In-Vitro Anti-Angiogenesis Activity of Anti-ROBO4 Antibody

The antibody of the present invention has an anti-angiogenesis activityin the absence of a cross-linking antibody in vitro. It is known thatcertain antibodies do not exhibit a pharmacological activity in theabsence of the cross-linking antibody in vitro, but exhibit apharmacological activity in the absence of the cross-linking antibody invivo (Cancer Cell (2011), 19, p. 101-113). This is probably becauseleukocytes are found in vivo to express Fcγ receptor having the samefunctions as those of the cross-linking antibody (Nature (2008), 8, p.34-47); thus, the antibodies exhibit a pharmacological activity throughcrosslink in the presence of leukocytes even without the cross-linkingantibody. In actual organisms, however, the number of leukocytes inlesions differs among individuals (Cancer Res (2011), 71, 5670-5677),presumably resulting in, among individuals, the different effects of theantibodies exhibiting a pharmacological activity dependent on crosslinkinduced by leucocytes. The antibody of the present invention exhibits anexcellent anti-angiogenesis activity even in the absence of across-linking antibody in vitro. Thus, the antibody of the presentinvention can also have an anti-angiogenesis effect independent of thenumber of leukocytes in vivo and is thus pharmaceutically suitable.

The anti-angiogenesis activity means the activity of suppressingvascular endothelial cell growth, migration, lumen formation, etc. Thein-vitro anti-angiogenesis activity can be evaluated by a vascularpermeability, vascular endothelial cell migration, or lumen formationtest.

For example, the vascular permeability test for such evaluation caninvolve inoculating a normal human umbilical vein endothelial cell(HUVEC) to the upper layer of Boyden Chamber having a pore size of 1 μmto form a single layer and then measuring the amount of FITC-labeleddextran or the like permeating through the cell layer. The amount ofFITC-labeled dextran permeating through the cell layer may be measuredusing, for example, In Vitro Vascular Permeability Assay (Cat. ECM640,manufactured by Millipore Corp.). When the antibody added at aconcentration of 5 μg/mL or lower exhibits the effect of suppressing theamount of FITC-labeled dextran permeating through the cell layer, thisantibody can be evaluated as having a suppressive effect on vascularpermeability and having an anti-angiogenesis activity. The antibody ofthe present invention exhibits a suppressive activity against vascularpermeability at a concentration of preferably 5 μg/mL or lower, morepreferably 1 μg/mL or lower, particularly preferably 0.5 μg/mL or lower,under the measurement conditions described above.

The cell migration test for such evaluation can involve inoculatingHUVEC to the upper layer of Boyden Chamber having a pore size of 3 to 8μm, adding a medium containing an endothelial cell migration enhancersuch as VEGF to the lower layer, and measuring the number of cellsmigrating to the lower layer. When the antibody exhibits the effect ofdecreasing the number of migrating HUVEC cells, this antibody can beevaluated as having a suppressive effect on vascular endothelial cellmigration and having an anti-angiogenesis activity. The number ofmigrating cells may be measured using, for example, vascular endothelialcell migration assay system (Cat. 354143, manufactured by BDBiosciences). The antibody of the present invention exhibits asuppressive activity against cell migration at a concentration ofpreferably 5 μg/mL or lower, more preferably 1 μg/mL or lower,particularly preferably 0.5 μg/mL or lower, under the measurementconditions described above.

The lumen formation test for such evaluation can involve inoculatingHUVEC to a cell culture container coated with Matrigel and measuring thenumber of branch points, tube length, or the like, of a lumen structureformed by HUVEC on the Matrigel. When the antibody exhibits the effectof decreasing the number of branch points or tube length of the lumenstructure, this antibody can evaluated as having a suppressive effect onlumen formation and having an anti-angiogenesis activity. The number ofbranch points or tube length of the lumen structure may be measuredusing, for example, vascular endothelial cell tube formation assaysystem (Cat. 354149, manufactured by BD Biosciences). The antibody ofthe present invention exhibits a suppressive activity against lumenformation at a concentration of preferably 5 μg/mL or lower, morepreferably 1 μg/mL or lower, particularly preferably 0.5 μg/mL or lower,under the measurement conditions described above.

Such an assay system, however, is not limited to these tests as long asit is capable of assaying angiogenesis and its suppression induced bythe ROBO4 protein.

The cross-linking antibody means an antibody that binds to the Fc regionof the antibody of the present invention and acts to cross-link two ormore antibody molecules of the present invention. For example, when theFc region of the antibody of the present invention is derived from amouse, the cross-linking antibody refers to an antibody that binds tothe mouse Fc region and associates two or more antibody molecules of thepresent invention through the binding of these two antibody molecules ofthe present invention at two binding sites, respectively, of thecross-linking antibody.

The phrase “having an anti-angiogenesis activity in the absence of across-linking antibody” means that the antibody exhibits ananti-angiogenesis effect in an evaluation system relating toangiogenesis suppression, for example, the evaluation system describedabove, even without coexisting with a cross-linking antibody.

The phrase “having an anti-angiogenesis activity in the presence of across-linking antibody” means that the antibody does not exhibit ananti-angiogenesis activity in the absence of a cross-linking antibody inan evaluation system relating to angiogenesis, for example, theanti-angiogenesis activity evaluation system described above, butexhibits the anti-angiogenesis activity when coexisting with one or morecross-linking antibody molecule(s), preferably two or more cross-linkingantibody molecules, with respect to one antibody molecule of the presentinvention.

(3-4) In-Vivo Suppressive or Inhibitory Activity of Anti-ROBO4 AntibodyAgainst Angiogenesis

The antibody of the present invention suppresses or inhibitsangiogenesis in vivo. The in-vivo suppressive or inhibitory activityagainst angiogenesis can be evaluated with animal disease modelsaccording to a standard method. For example, laser-induced choroidalneovascularization models described later in Example 4)-6 are widelyused as disease models of angiogenesis and can be used in evaluationwith the amount of blood vessels newly formed as a score. Also in thecase of patients, for example, tumor samples are collected by biopsyfrom tumor patients before and after administration of the antibody ofthe present invention, and the vascular densities of their intratumoralvessels can be measured by immunohistochemical analysis (IHC) to scorethe amount of blood vessels newly formed.

(3-5) Activation of Downstream Signal by Anti-ROBO4 Antibody

The anti-ROBO4 antibody of the present invention may be subjected to anevaluation system using a cell line or primary cultured cells thatexhibit some induced response to the ROBO4 protein. Examples of such acell line can include a mouse vascular endothelial cell line (ATCC NO.CRL-2779). Examples of such primary cultured cells can include mousevascular endothelial cells and human vascular endothelial cells.

The antibody of the present invention is an agonistic antibody againstROBO4. Specifically, the antibody of the present invention binds toROBO4 and activates the downstream signal of ROBO4. Thus, theanti-angiogenesis effect of the antibody of the present invention may beevaluated with the activation of the ROBO4 downstream signal as anindex. Examples of the ROBO4 downstream signal can include an IL-8promoter activity. The IL-8 promoter activity was drastically increasedin cells expressing full-length human ROBO4 compared with cellsexpressing no human ROBO4 and was hardly observed in cells expressingintracellular domain-deleted human ROBO4. Thus, the increase in IL-8promoter activity demonstrated that the activation of the ROBO4 signalwas detected (Example 3). The IL-8 promoter activity can be evaluated,for example, by the addition of the anti-ROBO4 antibody or theco-addition of the anti-ROBO4 antibody and a cross-linking antibody tocells transfected with a reporter vector having an IL-8 promotersequence insert and a human ROBO4 expression plasmid, followed by thedetermination of the reporter activity.

(3-6) Anti-ROBO4 Mouse Monoclonal Antibody and the Chimeric Antibody

MAb1 is an anti-ROBO4 mouse monoclonal antibody obtained by a methoddescribed in Example 2.

The nucleotide sequence of cDNA encoding the heavy chain variable regionof MAb1 is shown in SEQ ID NO: 30 (FIG. 15), and its amino acid sequenceis shown in SEQ ID NO: 31 (FIG. 16). The amino acid sequence of CDRH1 isshown in SEQ ID NO: 44 (FIG. 25); the amino acid sequence of CDRH2 isshown in SEQ ID NO: 46 (FIG. 26); and the amino acid sequence of CDRH3is shown in SEQ ID NO: 48 (FIG. 27). The nucleotide sequence of cDNAencoding the light chain variable region of MAb1 is shown in SEQ ID NO:32 (FIG. 17), and its amino acid sequence is shown in SEQ ID NO: 33(FIG. 18). The amino acid sequence of CDRL1 is shown in SEQ ID NO: 50(FIG. 28); the amino acid sequence of CDRL2 is shown in SEQ ID NO: 52(FIG. 29); and the amino acid sequence of CDRL3 is shown in SEQ ID NO:54 (FIG. 30).

The antibody mutant of the present invention, preferably, exhibitsreduced sensitivity to protein degradation or oxidation, an improvedbiological activity, an improved ability to bind to the antigen, orphysicochemical or functional properties imparted thereto, or the like.Examples of such an antibody mutant can include an antibody having anamino acid sequence derived from the amino acid sequence of the originalantibody by conservative amino acid substitution. The conservative aminoacid substitution is substitution that occurs in an amino acid grouprelated to amino acid side chains.

Preferable amino acid groups are as follows: an acidic group involvingaspartic acid and glutamic acid; a basic group involving lysine,arginine, and histidine; a nonpolar group involving alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan;and an uncharged polar family involving glycine, asparagine, glutamine,cysteine, serine, threonine, and tyrosine. Other preferable amino acidgroups are as follows: an aliphatic hydroxy group involving serine andthreonine; an amide-containing group involving asparagine and glutamine;an aliphatic group involving alanine, valine, leucine, and isoleucine;and an aromatic group involving phenylalanine, tryptophan, and tyrosine.Such amino acid substitution in the antibody mutant is preferablyperformed without reducing the antigen binding activity of the originalantibody.

An antibody mutant having an amino acid sequence derived from the aminoacid sequence of MAb1 of the present invention by conservative aminoacid substitution as well as a mouse antibody, rat antibody, chimericantibody, humanized antibody, human antibody, or the like comprising aCDR amino acid sequence derived from the amino acid sequence of any ofMAb1-derived CDRH1 to 3 and CDRL1 to 3 by conservative amino acidmutation is also encompassed by the present invention.

The constant regions of the antibody of the present invention are notparticularly limited. Preferably, those derived from a human antibodyare used in the antibody of the present invention for the treatment orprevention of a disease in a human. Examples of the heavy chain constantregion of the human antibody can include Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ,Cα1, Cα2, and Cε. Examples of the light chain constant region of thehuman antibody can include Cκ and Cλ.

A nucleotide sequence encoding the secretory signal-containing lightchain of cMAb1-1 exemplified as the mouse-human IgG1-type chimericantibody of the present invention and its amino acid sequence as well asa nucleotide sequence encoding the heavy chain thereof and its aminoacid sequence are shown in SEQ ID NOs: 37, 38, 39, and 40 (FIGS. 19, 20,21, and 22), respectively. Likewise, a nucleotide sequence encoding thesecretory signal-containing light chain of cMAb1-2 exemplified as themouse-human IgG2-type chimeric antibody of the present invention and itsamino acid sequence as well as a nucleotide sequence encoding the heavychain thereof and its amino acid sequence are shown in SEQ ID NOs: 37,38, 41, and 42 (FIGS. 19, 20, 23, and 24), respectively.

(3-7) Functional Fragment of Anti-ROBO4 Antibody

According to one aspect, the present invention provides a functionalfragment of the anti-ROBO4 antibody of the present invention. Thefunctional fragment of the antibody means a fragment maintaining atleast a portion of the functions of the antibody, or a modified formthereof described later in (3-10). Examples of such functions of theantibody can generally include an antigen binding activity, an antigenactivity-regulating activity, an antibody-dependent cytotoxic activity,and a complement-dependent cytotoxic activity. Examples of the functionsof the anti-ROBO4 antibody of the present invention can include a ROBO4protein binding activity, an anti-angiogenesis activity, and a ROBO4downstream signal-activating effect. More specifically, any functionalfragment having all or some of the above-described activities (3-3) to(3-5) exhibited by the antibody against ROBO4 of the present inventionis included in the functional fragment of the antibody of the presentinvention.

The functional fragment of the antibody is not particularly limited aslong as it is a fragment of the antibody maintaining at least a portionof the activities of the antibody, or a modified form thereof. Examplesthereof can include, but not limited to, Fab, F(ab′)2, Fv, single-chainFv (scFv) comprising heavy and light chain Fvs linked via an appropriatelinker, diabodies, linear antibodies, polyspecific antibodies formedfrom antibody fragments, and Fab′, which is a monovalent fragment ofantibody variable regions obtained by the treatment of F(ab′)2 underreducing conditions. A molecule containing a moiety other than thefragment of the antibody of the present invention, as in scFv carryingthe linker moiety, is also encompassed in the meaning of the functionalfragment of the antibody of the present invention.

A molecule that is derived from the antibody protein by the deletion of1 to several or more amino acid(s) at its amino terminus and/or carboxyterminus and maintains at least a portion of the functions of theantibody is also encompassed in the meaning of the functional fragmentof the antibody of the present invention.

The antibody of the present invention or the functional fragment thereofmay be a polyspecific antibody having specificity for at least 2 typesof different antigens. The polyspecific antibody is not limited to abispecific antibody, which binds to 2 types of different antigens, andan antibody having specificity for 3 or more types of different antigensis also encompassed in the meaning of the “polyspecific antibody” of thepresent invention.

The polyspecific antibody of the present invention may be a full-lengthantibody or a functional fragment thereof (e.g., bispecific F(ab′)2antibody). The bispecific antibody can also be prepared by binding theheavy and light chains (HL pairs) of two types of antibodies. Thebispecific antibody can also be obtained by fusing two or more types ofmonoclonal antibody-producing hybridomas to prepare bispecificantibody-producing fusion cells (Millstein et al., Nature (1983) 305, p.537-539). The polyspecific antibody can also be prepared in the same wayas above.

According to one aspect, the antibody of the present invention is asingle-chain antibody (single-chain Fv; hereinafter, referred to as“scFv”). The scFv is obtained by linking the heavy and light chainvariable regions of the antibody via a polypeptide linker (Pluckthun,The Pharmacology of Monoclonal Antibodies, 113, ed Rosenburg and Moore,Springer Verlag, New York, p. 269-315 (1994), Nature Biotechnology(2005), 23, p. 1126-1136). Moreover, bi-scFv comprising two scFvs linkedvia a polypeptide linker can be used as a bispecific antibody.Furthermore, multi-scFv comprising three or more scFvs can also be usedas a polyspecific antibody.

The present invention includes a single-chain immunoglobulin comprisingfull-length heavy and light chain sequences of the antibody linked viaan appropriate linker (Lee, H-S, et. al., Molecular Immunology (1999)36, p. 61-′71; Shirrmann, T. et. al., mAbs (2010), 2, (1) p. 1-4). Sucha single-chain immunoglobulin can be dimerized to thereby maintain astructure and activities similar to those of the antibody, which isoriginally a tetramer. Also, the antibody of the present invention maybe an antibody that has a single heavy chain variable region and has nolight chain sequence. Such an antibody is called a single domainantibody (sdAb) or a nanobody and has been reported to maintain theability to bind to the antigen (Muyldemans S. et. al., Protein Eng.(1994) 7 (9), 1129-35, Hamers-Casterman C. et. al., Nature (1993) 363(6428) 446-8). These antibodies are also encompassed in the meaning ofthe functional fragment of the antibody according to the presentinvention.

(3-8) Anti-Human ROBO4 Humanized Antibody (Hereinafter “Anti-ROBO4Humanized Antibody”)

According to one aspect, the present invention provides a humanizedantibody or a functional fragment thereof. The anti-ROBO4 humanizedantibody of the present invention or the functional fragment thereof hasan anti-angiogenesis activity and, preferably, has an anti-angiogenesisactivity in vivo. Preferably, the humanized antibody or the functionalfragment thereof specifically binds to the ROBO4 protein. Moreover, thehumanized antibody or the functional fragment thereof is an agonisticantibody against ROBO4 and activates its downstream signal. Furthermore,the humanized antibody or the functional fragment thereof suppresses orinhibits vascular endothelial cell migration in the absence of across-linking antibody in vitro.

Examples of the humanized antibody of the present invention can includea human-derived antibody having MAb1 complementarity determining regions(CDRs) replaced with the CDRs of a non-human animal antibody (see Nature(1986) 321, p. 522-525), and a human antibody grafted with the CDRsequences and with some amino acid residues of framework regions by aCDR grafting method (International Publication No. WO90/07861).Furthermore, a variant derived from the humanized antibody by thesubstitution of 1 to 3 amino acid residues in each CDR with other aminoacid residues is also included in the antibody of the present inventionas long as the variant has all or some of the activities (3-3) to (3-5).

Preferred examples of the anti-ROBO4 humanized antibody of the presentinvention or the functional fragment thereof can include an antibodythat consists of a heavy chain having a variable region comprising CDRH1consisting of the amino acid sequence represented by SEQ ID NO: 44 (FIG.25), CDRH2 consisting of the amino acid sequence represented by SEQ IDNO: 46 (FIG. 26) or an amino acid sequence derived from the amino acidsequence of SEQ ID NO: 46 by the substitution of one amino acid, andCDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 48(FIG. 27), and a light chain having a variable region comprising CDRL1consisting of the amino acid sequence represented by SEQ ID NO: 50 (FIG.28) or an amino acid sequence derived from the amino acid sequence ofSEQ ID NO: 50 by the substitution of 1 to 3 amino acid(s), CDRL2consisting of the amino acid sequence represented by SEQ ID NO: 52 (FIG.29), and CDRL3 consisting of the amino acid sequence represented by SEQID NO: 54 (FIG. 30), and recognizes the ROBO4 protein of the presentinvention, and a fraction of the antibody maintaining the ROBO4 proteinbinding activity of the antibody.

Examples of the amino acid substitution in CDRH2 can include thesubstitution of the amino acid represented by amino acid No. 4 of SEQ IDNO: 46 in CDRH2. Specifically, an asparagine, amino acid No. 4 of SEQ IDNO: 46, can be replaced with a glutamine. The amino acid to besubstituted therefor is not limited as long as the resulting antibodyhas all or some of the activities (3-3) to (3-5) exhibited by theantibody against ROBO4 of the present invention.

Examples of the amino acid substitution in CDRL1 can include thesubstitution of any 1 to 3, preferably 3, of the amino acids representedby amino acid Nos. 9, 11, and 13 of SEQ ID NO: 50 in CDRL1.Specifically, a serine (amino acid No. 9), a glycine (amino acid No. 11)and a threonine (amino acid No. 13) of SEQ ID NO: 50 can be replacedwith an amino acid selected from a glutamic acid, a lysine and aleucine, preferably with a glutamic acid, a lysine and a leucinerespectively. The amino acid(s) to be substituted therefor is notlimited as long as the resulting antibody has all or some of theactivities (3-3) to (3-5) exhibited by the antibody against ROBO4 of thepresent invention.

An asparagine residue in peptides or a protein is reported to easilyundergo deamidation in some conditions (Gerger et al: The Journal ofBiological Chemistry Vol. 262 No. 2, 785-794, 1987), therefore the aminoacid replacement in CDRs described above can increase the stability ofthe humanized antibodies of the present invention.

Examples of the heavy chain variable region of the more preferredhumanized antibody having these CDRHs can include an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 56 (FIG. 32),wherein CDRH1, CDRH2 and CDRH3 are represented by amino acid Nos. 50 to54, 69 to 85 and 118 to 126 of SEQ ID NO: 56 (FIG. 32), respectively, anamino acid sequence represented by amino acid Nos. 20 to 137 of SEQ IDNO: 58 (FIG. 34), wherein CDRH1, CDRH2 and CDRH3 are represented byamino acid Nos. 50 to 54, 69 to 85 and 118 to 126 of SEQ ID NO: 58 (FIG.34), respectively, an amino acid sequence represented by amino acid Nos.20 to 137 of SEQ ID NO: 60 (FIG. 36), wherein CDRH1, CDRH2 and CDRH3 arerepresented by amino acid Nos. 50 to 54, 69 to 85 and 118 to 126 of SEQID NO: 60 (FIG. 36), respectively, and an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 62 (FIG. 38),wherein CDRH1, CDRH2 and CDRH3 are represented by amino acid Nos. 50 to54, 69 to 85 and 118 to 126 of SEQ ID NO: 60 (FIG. 38). Examples of thelight chain variable region of the more preferred humanized antibodyhaving these CDRLs can include an amino acid sequence represented byamino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40), wherein CDRL1,CDRL2 and CDRL3 are represented by amino acid Nos. 44 to 59, 75 to 81and 114 to 122 of SEQ ID NO: 64 (FIG. 40), respectively, and an aminoacid sequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 66(FIG. 42), wherein CDRL1, CDRL2 and CDRL3 are represented by amino acidNos. 44 to 59, 75 to 81 and 114 to 122 of SEQ ID NO: 66 (FIG. 42),respectively.

Examples of more preferable combinations of the heavy chain variableregion and the light chain variable region of the more preferredhumanized antibodies can include: a humanized antibody comprising aheavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 58 (FIG. 34) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40); ahumanized antibody comprising a heavy chain variable region consistingof an amino acid sequence represented by amino acid Nos. 20 to 137 ofSEQ ID NO: 58 (FIG. 34) and a light chain variable region consisting ofan amino acid sequence represented by amino acid Nos. 21 to 134 of SEQID NO: 66 (FIG. 42); a humanized antibody comprising a heavy chainvariable region consisting of an amino acid sequence represented byamino acid Nos. 20 to 137 of SEQ ID NO: 62 (FIG. 38) and a light chainvariable region consisting of an amino acid sequence represented byamino acid Nos. 21 to 134 of SEQ ID NO: 66 (FIG. 42); a humanizedantibody comprising a heavy chain variable region consisting of an aminoacid sequence represented by amino acid Nos. 20 to 137 of SEQ ID NO: 62(FIG. 38) and a light chain variable region consisting of an amino acidsequence represented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG.40); a humanized antibody comprising a heavy chain variable regionconsisting of an amino acid sequence represented by amino acid Nos. 20to 137 of SEQ ID NO: 56 (FIG. 32) and a light chain variable regionconsisting of an amino acid sequence represented by amino acid Nos. 21to 134 of SEQ ID NO: 64 (FIG. 40); and a humanized antibody comprising aheavy chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 20 to 137 of SEQ ID NO: 60 (FIG. 36) anda light chain variable region consisting of an amino acid sequencerepresented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 (FIG. 40).

Even more preferred examples of the full-length humanized antibodycomprising the more preferred combination of the heavy chain variableregion and the light chain variable region can include: a humanizedantibody (H-1140) comprising a heavy chain consisting of an amino acidsequence represented by amino acid Nos. 20 to 463 of SEQ ID NO: 58 (FIG.34) and a light chain consisting of an amino acid sequence representedby amino acid Nos. 21 to 239 of SEQ ID NO: 64 (FIG. 40); a humanizedantibody (H-1143) comprising a heavy chain consisting of an amino acidsequence represented by amino acid Nos. 20 to 463 of SEQ ID NO: 58 (FIG.34) and a light chain consisting of an amino acid sequence representedby amino acid Nos. 21 to 239 of SEQ ID NO: 66 (FIG. 42); a humanizedantibody (H-2143) comprising a heavy chain consisting of an amino acidsequence represented by amino acid Nos. 20 to 463 of SEQ ID NO: 62 (FIG.38) and a light chain consisting of an amino acid sequence representedby amino acid Nos. 21 to 239 of SEQ ID NO: 66 (FIG. 42); a humanizedantibody (H-2140) comprising a heavy chain consisting of an amino acidsequence represented by amino acid Nos. 20 to 463 of SEQ ID NO: 62 (FIG.38) and a light chain consisting of an amino acid sequence representedby amino acid Nos. 21 to 239 of SEQ ID NO: 64 (FIG. 40); a humanizedantibody (H-1040) comprising a heavy chain consisting of an amino acidsequence represented by amino acid Nos. 20 to 463 of SEQ ID NO: 56 (FIG.32) and a light chain consisting of an amino acid sequence representedby amino acid Nos. 21 to 239 of SEQ ID NO: 64 (FIG. 40); and a humanizedantibody (H-2040) comprising a heavy chain consisting of an amino acidsequence represented by amino acid Nos. 20 to 463 of SEQ ID NO: 60 (FIG.36) and a light chain consisting of an amino acid sequence representedby amino acid Nos. 21 to 239 of SEQ ID NO: 64 (FIG. 40).

The most preferable antibodies of the present invention are H-1140,H-1143, H-2140 and H-2143.

H-1140 has properties of 1) binding specifically to human ROBO4 and notto ROBO1, ROBO2, and ROBO3, 2) having a K_(D) value of 3.9 nM for humanROBO4, 3) maintaining affinity to human ROBO4 under 40° C. for 4 weeks,4) inhibiting HUVEC migration induced by one or more angiogenic factorsselected from VEGF, bFGF, HGF, PDGF-BB and SDF-1, specifically VEGF orbFGF, 5) inhibiting angiogenesis in vivo, and 6) low immunogenicity inISPRI Web-based Immunogenicity Screening (EpiVax, Inc.).

H-1143 has properties of 1) binding specifically to human ROBO4 and notto ROBO1, ROBO2, and ROBO3, 2) having a K_(D) value of 3.5 nM for humanROBO4, 3) maintaining affinity to human ROBO4 under 40° C. for 4 weeks,4) inhibiting HUVEC migration induced by one or more angiogenic factorsselected from VEGF, bFGF, HGF, PDGF-BB and SDF-1, specifically VEGF andbFGF, 5) inhibiting angiogenesis in vivo, and 6) low immunogenicity inEpiScreen™ immunogenicity testing (Antitope Ltd.)

H-2140 has properties of 1) binding specifically to human ROBO4 and notto ROBO1, ROBO2, and ROBO3, 2) having a K_(D) value of 1.8 nM for humanROBO4, 3) maintaining affinity to human ROBO4 under 40° C. for 4 weeks,4) inhibiting HUVEC migration induced by various angiogenic factors suchas VEGF, bFGF, HGF, PDGF-BB and SDF-1, specifically VEGF and bFGF, 5)inhibiting angiogenesis in vivo, 6) low immunogenicity in EpiScreen™immunogenicity testing (Antitope Ltd.)

H-2143 has properties of 1) binding specifically to human ROBO4 and notto ROBO1, ROBO2, and ROBO3, 2) having a K_(D) value of 1.7 nM for humanROBO4, 3) maintaining affinity to human ROBO4 under 40° C. for 4 weeks,4) inhibiting HUVEC migration induced by various angiogenic factors suchas VEGF, bFGF, HGF, PDGF-BB and SDF-1, specifically VEGF and bFGF, 5)inhibiting angiogenesis in vivo, 6) low immunogenicity in EpiScreen™immunogenicity testing (Antitope Ltd.), and 7) showing no serious changein clinical sign, body weight, food consumption, hematology, bloodchemistry, pathology, electroretinography after single intravitrealinjection (2.75 mg/eye) to a Cynomolgus monkey.

An antibody comprising an amino acid sequence having 95% or more,preferably 97% or more, more preferably 99% or more identity to theamino acid sequence of the antibody such as H-1140, H-1143, H-2143,H-2140, H-1040 and H-2040 is also included in the antibody of thepresent invention as long as the antibody has all or some of theactivities (3-3) to (3-5). Moreover, an antibody that has CDRs identicalin amino acid sequence to the CDRs of the antibody comprising thecombination of the heavy chain variable region and the light chainvariable region or the antibody comprising the combination of the heavychain and the light chain, and has an amino acid sequence other than theCDR amino acid sequence having 95% or more, preferably 97% or more, morepreferably 99% or more identity thereto is also included in the antibodyof the present invention as long as the antibody has all or some of theactivities (3-3) to (3-5).

(3-9) Antibody Binding to the Same Site

An “antibody binding to the same site” as that bound by the antibodyprovided by the present invention is also included in the antibody ofthe present invention. The “antibody binding to the same site” as thatbound by a certain antibody means another antibody that binds to a siteon an antigen molecule recognized by the antibody. If a second antibodybinds to a partial peptide or a partial three-dimensional structure onan antigen molecule bound by a first antibody, the first and secondantibodies can be determined to bind to the same site. Moreover, thefirst and second antibodies can be determined to bind to the same siteby confirming that the second antibody competes with the first antibodyfor binding to the antigen, i.e., the second antibody interferes withthe binding of the first antibody to the antigen, even if the peptidesequence or three-dimensional structure of the specific binding site isnot determined. Furthermore, when the first and second antibodies bindto the same site and the first antibody has an effect characteristic ofone aspect of the antibody of the present invention, such as ananti-angiogenesis activity, the second antibody also has an exceedinglyhigh probability of having the same activity thereas. Thus, if a secondanti-ROBO4 antibody binds to the site bound by a first anti-ROBO4antibody, the first and second antibodies can be determined to bind tothe same site on the ROBO4 protein. Moreover, the first and secondantibodies can be determined to be antibodies binding to the same siteon the ROBO4 protein by confirming that the second anti-ROBO4 antibodycompetes with the first anti-ROBO4 antibody for binding to the ROBO4protein.

An antibody binding to a site on the ROBO4 protein recognized by MAb1 ofthe present invention is also included in the present invention.

The antibody-binding site can be determined by a method well known bythose skilled in the art, such as immunoassay. For example, a series ofpeptides are prepared by the appropriate C-terminal or N-terminaltruncation of the amino acid sequence of the antigen, and the reactivityof the antibody thereto is studied to roughly determine a recognitionsite. Then, shorter peptides are synthesized, and the reactivity of theantibody to these peptides can be studied to thereby determine thebinding site. The antigen fragment peptides can be prepared using atechnique such as gene recombination or peptide synthesis.

When the antibody binds to or recognizes the partial conformation of theantigen, the binding site for the antibody can be determined byidentifying amino acid residues on the antigen adjacent to the antibodyusing an X-ray structural analysis.

(3-10) Modified Form of Anti-ROBO4 Antibody or Functional FragmentThereof

The present invention provides a modified form of the antibody or thefunctional fragment thereof. The modified form of the antibody of thepresent invention or the functional fragment thereof means an antibodyof the present invention or a functional fragment thereof provided withchemical or biological modification. The chemically modified formincludes a form having an amino acid skeleton conjugated with a chemicalmoiety, a form having a chemically modified N-linked or O-linkedcarbohydrate chain, and the like. Said chemical moiety or form can betoxic or cytotoxic.

The biologically modified form includes a form that has undergonepost-translational modification (e.g., N-linked or O-linkedglycosylation, N-terminal or C-terminal processing, deamidation,isomerization of aspartic acid, or oxidation of methionine), a formcontaining a methionine residue added to the N-terminus by expressionusing prokaryotic host cells, and the like. Such a modified form is alsomeant to include a form labeled to permit detection or isolation of theantibody or the antigen of the present invention, for example, anenzyme-labeled form, a fluorescently labeled form, or anaffinity-labeled form. Such a modified form of the antibody of thepresent invention or the functional fragment thereof is useful inimprovement in the stability or blood retention of the original antibodyof the present invention or functional fragment thereof, reduction inantigenicity, detection or isolation of the antibody or the antigen,etc.

Examples of the chemical moiety contained in the chemically modifiedform can include water-soluble polymers such as polyethylene glycol,ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,dextran, and polyvinyl alcohol.

Examples of the biologically modified form can include a form modifiedby enzymatic treatment, cell treatment, or the like, a form fused withanother peptide, such as a tag, added by gene recombination, and a formprepared from host cells expressing an endogenous or exogenous sugarchain-modifying enzyme.

Such a modification may be made at an arbitrary position or the desiredposition in the antibody or the functional fragment thereof.Alternatively, the same or two or more different modifications may bemade at one or two or more positions therein.

In the present invention, the “modified form of the antibody fragment”is also meant to include even a “fragment of the modified form of theantibody”.

For example, occasionally, an antibody produced by cultured mammaliancells is known to lack a carboxyl-terminal lysine residue in its heavychain (Journal of Chromatography A, 705: 129-134 (1995)). It is alsoknown that occasionally 2 carboxyl-terminal amino acid residues (i.e.,glycine and lysine) of a heavy chain are missing and that a prolineresidue newly located at the carboxyl terminus is amidated (AnalyticalBiochemistry, 360: 75-83 (2007)). Such lack or modification in theseheavy chain sequences, however, affects neither the ability of theantibody to bind to its antigen nor the effector functions (complementactivation, antibody-dependent cytotoxicity, etc.) of the antibody.Thus, an antibody having the modification and a functional fragment ofthe antibody are also included in the antibody of the present invention.Examples of such an antibody can include a deletion mutant derived fromthe antibody of the present invention by the deletion or lack of 1 or 2amino acid(s) in the carboxyl terminus of the heavy chain, and thedeletion mutant having an amidated residue (e.g., an amidated prolineresidue at the carboxyl-terminal site of the heavy chain). However, thedeletion mutant of the antibody according to the present invention isnot limited to the types described above as long as the deletion mutantmaintains the ability to bind to the antigen and all or some of theactivities (3-3) to (3-5). Two heavy chains constituting the antibodyaccording to the present invention may be composed of any one type ofheavy chain selected from the group consisting of the full-length heavychains and the heavy chains of the deletion mutant or may be composed ofthe combination of any two types selected therefrom. The quantitativeratio of the deletion variant heavy chain(s) is susceptible to, forexample, the type of cultured mammalian cells producing the antibodyaccording to the present invention, and the culture conditions of thecells. Examples of such deletion variant heavy chains as the maincomponents of the antibody according to the present invention caninclude two heavy chains, both of which lack one carboxyl-terminal aminoacid residue. All of these deletion variants are encompassed in theantibody variant, the functional fragment of the antibody, or themodified form thereof according to the present invention.

4. Method for Producing Antibody

(4-1) Method Using Hybridoma

In order to prepare the anti-ROBO4 antibody of the present invention,anti-ROBO4 antibody-producing cells are isolated from the spleens ofanimals immunized with the ROBO4 protein according to the method ofKohler and Milstein (Kohler and Milstein, Nature (1975) 256, p. 495-497,Kennet, R. ed., Monoclonal Antibody, p. 365-367, Prenum Press, N.Y.(1980)). The cells are fused with myeloma cells to thereby establishhybridomas, and monoclonal antibodies can be obtained from cultures ofthese hybridomas.

(4-1-1) Preparation of Antigen

The antigen for the preparation of the anti-ROBO4 antibody can beobtained according to, for example, a native or recombinant ROBO4protein preparation method described in other paragraphs of the presentspecification. Examples of the antigen that may be thus prepared caninclude the ROBO4 protein or a ROBO4 protein fragment comprising apartial sequence with at least 6 consecutive amino acids thereof, andtheir derivatives further comprising an arbitrary amino acid sequence orcarrier added thereto (hereinafter, collectively referred to as a “ROBO4antigen”).

The recombinant ROBO4 antigen can be prepared by transfecting host cellswith a gene comprising a nucleotide sequence encoding the amino acidsequence of the ROBO4 antigen, and collecting the antigen from culturesof the cells. The native ROBO4 antigen can be purified or isolated from,for example, human or rodent tissues with angiogenesis, cells derivedfrom the tissues, or cultures of the cells. A ROBO4 antigen obtained ina cell-free in-vitro translation system from a gene comprising anucleotide sequence encoding the amino acid sequence of the ROBO4antigen is also included in the “ROBO4 antigen” of the presentinvention.

(4-1-2) Production of Anti-ROBO4 Monoclonal Antibody

The monoclonal antibody is typically produced by the following steps:

(a) preparing an antigen,

(b) preparing antibody-producing cells,

(c) preparing myeloma cells (hereinafter, referred to as “myelomas”),

(d) fusing the antibody-producing cells with the myelomas,

(e) screening for a hybridoma group producing the antibody of interest,and

(f) obtaining single cell clones (cloning).

This production method further involves (g) a step of culturing thehybridomas, a step of raising hybridoma-transplanted animals, etc., (h)a step of assaying or determining the biological activity of themonoclonal antibody, etc., if necessary.

Hereinafter, the method for preparing the monoclonal antibody will bedescribed in detail with reference to these steps. However, the methodfor preparing the antibody is not limited to them, and, for example,antibody-producing cells other than spleen cells and myelomas may beused.

(a) Step of Preparing Antigen

An antigen can be prepared according to the ROBO4 protein preparationmethod described above in (2-3).

(b) Step of Preparing Antibody-Producing Cells

The antigen obtained in step (a) is mixed with an adjuvant such as aFreund's complete or incomplete adjuvant or potassium aluminum sulfate,and laboratory animals are immunized with the resulting immunogen. Anylaboratory animal used in a hybridoma preparation method known in theart can be used without limitations. Specifically, for example, mice,rats, goats, sheep, cattle, or horses can be used. From the viewpoint ofreadily available myeloma cells to be fused with isolatedantibody-producing cells, etc., the animals to be immunized arepreferably mice or rats.

Mouse and rat strains actually used are not particularly limited. In thecase of mice, for example, A, AKR, BALB/c, BALB/cAnNCrj, BDP, BA, CE,C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HT1, LP, NZB, NZW, RF, R III, SJL,SWR, WB, 129 can be used. In the case of rats, for example, Wistar, Low,Lewis, Sprague-Dawley, ACI, BN, or Fischer can be used.

These mice and rats can be available from laboratory animalbreeders/distributors, for example, CLEA Japan, Inc. or Charles RiverLaboratories Japan Inc.

Of them, a BALB/c mouse strain or Wistar and Low rat strains areparticularly preferable as animals to be immunized in consideration offusion compatibility with the myeloma cells described later.

Also in consideration of the homology between human and mouse antigens,mice whose biological mechanism to remove autoantibodies has beenreduced, i.e., autoimmune disease mice, is also preferably used.

In this context, these mice or rats are preferably 5 to 12 weeks old,more preferably 6 to 8 weeks old at the time of immunization.

The animals can be immunized with the ROBO4 protein using, for example,the method of Weir, D. M., Handbook of Experimental Immunology Vol. I.II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A.and Mayer, M. M., Experimental Immunochemistry, Charles C ThomasPublisher Spigfield, Ill. (1964).

Examples of antibody titer determination methods can include, but notlimited to, immunoassay such as MA and ELISA.

Antibody-producing cells derived from spleen cells or lymphocytesseparated from the immunized animals can be prepared according to amethod known in the art, for example, the method of Kohler et al.,Nature (1975) 256, p. 495; Kohler et al., Eur. J. Immnol. (1977) 6, p.511, Milstein et al., Nature (1977), 266, p. 550; Walsh, Nature, (1977)266, p. 495,).

In the case of spleen cells, a general method can be adopted, whichinvolves chopping the spleens, filtering cells through a stainless mesh,and then floating the resulting cells in an Eagle's minimum essentialmedium (MEM) or the like to separate antibody-producing cells.

(c) Step of Preparing Myelomas

The myeloma cells used in cell fusion are not particularly limited andcan be selected appropriately for use from cell lines known in the art.An HGPRT (hypoxanthine-guanine phosphoribosyl transferase)-deficientline, i.e., mouse-derived X63-Ag8 (X63), NS1-ANS/1 (NS1), P3X63-Ag8.U1(P3U1), X63-Ag8.653 (X63.653), SP2/0-Ag14 (SP2/0), MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO, BU.1 or the like, rat-derived 210.RSY3.Ag.1.2.3(Y3) or the like, or human-derived U266AR (SKO-007), GM1500-GTG-A12(GM1500), UC729-6, LICR-LOW-HMy2 (HMy2), 8226AR/NIP4-1 (NP41), or thelike, whose screening procedures have already been established, ispreferably used in consideration of convenience in the selection ofhybridomas from fusion cells. These HGPRT-deficient lines can beavailable from, for example, American Type Culture Collection (ATCC).

These cell lines are subcultured in an appropriate medium, for example,an 8-azaguanine medium [RPMI-1640 medium supplemented with glutamine,2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter,referred to as “FCS”) and further supplemented with 8-azaguanine], anIscove's modified Dulbecco's medium (hereinafter, referred to as“IMDM”), or a Dulbecco's modified Eagle medium (hereinafter, referred toas “DMEM”) and subcultured 3 to 4 days before cell fusion in a normalmedium [e.g., ASF104 medium (manufactured by Ajinomoto Co., Inc.)containing 10% FCS] to secure the number of cells equal to or largerthan 2×10⁷ cells on the day of cell fusion.

(d) Step of Fusing the Antibody-Producing Cells with the Myeloma Cells

The antibody-producing cells can be fused with the myeloma cells underconditions that prevent cell viability from being exceedingly reduced,according to a method known in the art (Weir, D. M., Handbook ofExperimental Immunology Vol. I. II. III., Blackwell ScientificPublications, Oxford (1987), Kabat, E. A. and Mayer, M. M., ExperimentalImmunochemistry, Charles C Thomas Publisher Spigfield, Ill. (1964)etc.). For example, a chemical method which involves mixingantibody-producing cells with myeloma cells in a high-concentrationsolution of a polymer such as polyethylene glycol, or a physical methodusing electric stimulation can be used.

(e) Step of Screening for a Hybridoma Group Producing the Antibody ofInterest

A selection method for the hybridomas obtained by cell fusion is notparticularly limited, and HAT (hypoxanthine-aminopterin-thymidine)selection method (Kohler et al., Nature (1975) 256, p. 495; Milstein etal., Nature (1977) 266, p. 550) is typically used. This method iseffective for obtaining hybridomas using an HGPRT-deficient myeloma cellline, which cannot survive in the presence of aminopterin. Specifically,unfused cells and hybridomas can be cultured in a HAT medium to therebyallow only hybridomas resistant to aminopterin to selectively remain andgrow.

(f) Step of Obtaining Single Cell Clones (Cloning)

The hybridomas can be cloned using a method known in the art, forexample, a methylcellulose, soft agarose, or limiting dilution method(see e.g., Barbara, B. M. and Stanley, M. S.: Selected Methods inCellular Immunology, W. H. Freeman and Company, San Francisco (1980)).The limiting dilution method is preferable.

(g) Step of Culturing the Hybridomas and Step of RaisingHybridoma-Transplanted Animals

The selected hybridomas can be cultured to thereby produce monoclonalantibodies. Preferably, the desired hybridomas are cloned and thensubjected to antibody production.

The monoclonal antibody produced by this hybridoma can be collected fromcultures of the hybridoma. Moreover, a recombinant antibody can also becollected from cultures of cells transfected with the monoclonalantibody gene. Furthermore, the hybridomas can be injectedintraperitoneally to mice of the same strain (e.g., BALB/cAnNCrjdescribed above) or Nu/Nu mice and allowed to grow. Then, the monoclonalantibody can also be collected from their ascites.

(h) Step of Assaying or Determining the Biological Activity of theMonoclonal Antibody

Various biological tests can be selected and applied thereto accordingto the purpose.

(4-2) Cell Immunization Method

Cells expressing the native ROBO4 protein, cells expressing therecombinant ROBO4 protein or its fragment, or the like can be used asimmunogens to thereby prepare an anti-ROBO4 antibody by the hybridomamethod described above.

Examples of the cells expressing the native ROBO4 protein can includecells derived from patients affected with an angiogenic disease such asproliferative diabetic retinopathy or tumor, and cells derived from thetissues of these patients. Such cells are preferably vascularendothelial cells, but not limited to them. These ROBO4protein-expressing cells are used in an amount of 1×10⁵ to 1×10⁹ cells,preferably 1×10⁶ to 1×10⁸ cells, more preferably 0.5 to 2×10⁷ cells,even more preferably 1×10⁷ cells, in one immunization. The number ofcells subjected to immunization can be changed according to theexpression level of the ROBO4 protein. The immunogens are generallyadministered intraperitoneally and may be administered through anintradermal route or the like. The method described in (4-1-2) can beapplied to the hybridoma preparation approach.

(4-3) Gene Recombination and Host Cells

In order to prepare the antibody of the present invention, host cellsare transfected with a nucleotide comprising a nucleotide sequenceencoding the amino acid sequence of its heavy chain (heavy chainnucleotide) and a nucleotide comprising a nucleotide sequence encodingthe amino acid sequence of its light chain (light chain nucleotide), orwith a vector containing an insert of the heavy chain nucleotide and avector containing an insert of the light chain nucleotide, and thencultured, and the antibody can be collected from the cultures. The heavyand light chain nucleotides may be inserted in one vector.

Prokaryotic or eukaryotic cells can be used as host cells. Wheneukaryotic cells are used as hosts, animal cells, plant cells, oreukaryotic microbes can be used.

Examples of the animal cells can include mammal-derived cells, i.e.,monkey-derived COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCCCRL-1650), mouse fibroblast NIH3T3 (ATCC No.CRL-1658), mouse NS0 celllines (ECACC), Chinese hamster ovary cells (CHO cells, ATCC CCL-61),dihydrofolate reductase-deficient lines thereof (CHOdhfr−: Urlaub, G.and Chasin, L. A. Proc. Natl. Acad. Sci. U.S.A. (1980) 77, p.4126-4220), CHOK1SV developed by Lonza Biologics, cells derived frombirds such as chickens, and cells derived from insects.

Also, host cells of the present invention include cells that may producean antibody protein, wherein the structure of a sugar chain attached tothe antibody protein is modified, wherein a biological activity of theantibody with the modification is preferably enhanced compared to theantibody without the modification. Example of such host cells of thepresent invention includes CHO cells that may produce an antibodyprotein having complex N-glycoside-linked sugar chains bound to the Fcregion of the antibody, wherein among the total complexN-glycoside-linked sugar chains bound to the Fc region of the antibody,the ratio of a sugar chain in which fucose is not bound toN-acetylglucosamine in the reducing end in the sugar chain is 20% ormore (WO2000/61739, WO2002/31140).

Examples of the eukaryotic microbes can include yeasts. Examples of theprokaryotic cells can include E. coli and Bacillus subtilis.

Mammal-derived cells are preferably used, CHO cells are more preferablyused, and CHOK1SV are even more preferably used as host cells forproducing the anti-ROBO4 antibodies of the present invention.

A signal peptide for the secretion of the antibody of the presentinvention (monoclonal antibodies derived from various animal species,rat antibody, mouse antibody, chimeric antibody, humanized antibody,human antibody, etc.) is not limited to the secretory signal of anantibody of the same species, the same type, and the same subtype as theantibody or to the secretory signal of the antibody itself. Anysecretory signal of an antibody of different type or subtype therefromor any secretory signal of a protein derived from a different eukaryoticspecies therefrom or a prokaryotic species can be selected and used.

(4-4) Methods for Designing and Preparing a Humanized Antibody

Examples of the humanized antibody can include, but not limited to, ahuman-derived antibody having CDRs replaced with the CDRs of a non-humananimal antibody (see Nature (1986) 321, p. 522-525), a human antibodygrafted with the CDR sequences and with some amino acid residues offramework regions by a CDR grafting method (see WO90/07861 and U.S. Pat.No. 6,972,323), and an antibody having human antibody amino acid(s)replaced for one or two or more non-human animal antibody-derived aminoacid(s) in any of these humanized antibodies.

(4-5) Method for Preparing a Human Antibody

Further examples of the antibody of the present invention can include ahuman antibody. The anti-ROBO4 human antibody means an anti-ROBO4antibody consisting of the amino acid sequence of a human-derivedantibody. The anti-ROBO4 human antibody can be obtained by a methodusing human antibody-producing mice carrying a human genomic DNAfragment comprising human antibody heavy chain- and light chain-encodinggenes (see Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143;Kuroiwa, Y. et. al., Nuc. Acids Res. (1998) 26, p. 3447-3448; Yoshida,H. et. al., Animal Cell Technology: Basic and Applied Aspects vol. 10,p. 69-73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.), Kluwer AcademicPublishers, 1999; Tomizuka, K. et. al., Proc. Natl. Acad. Sci. USA(2000) 97, p. 722-727 etc.).

Specifically, such human antibody-producing animals can be prepared bydisrupting the endogenous immunoglobulin heavy and light chain gene lociof non-human mammals and instead introducing thereto humanimmunoglobulin heavy and light chain gene loci via yeast artificialchromosome (YAC) vectors or the like. Alternatively, eukaryotic cellsare transformed with heavy chain- and light chain-encoding cDNAs of sucha human antibody, preferably with vectors comprising each of the cDNAs,by a gene recombination technique, and the transformed cells producing arecombinant human monoclonal antibody are cultured. This antibody can beobtained from the culture supernatant.

In this context, for example, eukaryotic cells, preferably mammaliancells such as CHO cells, lymphocytes, or myelomas can be used as hosts.

Also, a method for obtaining a phage display-derived human antibodyselected from a human antibody library (see Wormstone, I. M. et. al,Investigative Ophthalmology & Visual Science. (2002) 43 (7), p.2301-2308; Carmen, S. et. al., Briefings in Functional Genomics andProteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. al.,Opthalmology (2002) 109 (3), p. 427-431 etc.) is known.

For example, a phage display method (Nature Biotechnology (2005), 23,(9), p. 1105-1116) can be used, which involves allowing the variableregions of a human antibody to be expressed as a single-chain antibody(scFv) on phage surface and selecting a phage binding to the antigen.

The phage selected based on its binding to the antigen can be subjectedto gene analysis to thereby determine DNA sequences encoding thevariable regions of the human antibody binding to the antigen.

If the DNA sequence of scFv binding to the antigen is determined, anexpression vector having this sequence is prepared and appropriate hostscan be transfected with the expression vector and allowed to express thehuman antibody (WO92/01047, WO92/20791, WO93/06213, WO93/11236,WO93/19172, WO95/01438, WO95/15388, Annu. Rev. Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116.

(4-6) Method for Preparing Functional Fragments of an Antibody

The method for preparing a single-chain antibody is well known in theart (see e.g., U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, and5,455,030). In this scFv, a heavy chain variable region and a lightchain variable region are linked via a linker that prevents them fromforming a conjugate, preferably a polypeptide linker (Huston, J. S. etal., Proc. Natl. Acad. Sci. U.S.A. (1988), 85, p. 5879-5883). The heavychain variable region and the light chain variable region in scFv may bederived from the same antibody or may be derived from differentantibodies.

For example, an arbitrary single-chain peptide consisting of 12 to 19residues is used as the polypeptide linker that links these variableregions.

In order to obtain scFv-encoding DNA, of the sequences of DNA encodingthe heavy chain or heavy chain variable region of the antibody and DNAencoding the light chain or light chain variable region thereof, eachDNA portion encoding the whole or desired amino acid sequence is used asa template and amplified by PCR using a primer pair flanking both endsof the template. Subsequently, DNA encoding the polypeptide linkermoiety is further amplified in combination with a primer pair flankingboth ends thereof to link them to the heavy and light chains,respectively.

The scFv-encoding DNA can be used to thereby prepare, according to aroutine method, an expression vector containing the DNA and host cellstransformed with the expression vector. In addition, the host cells arecultured, and the scFv can be collected from the cultures according to aroutine method.

Also in order to obtain other functional fragments of the antibody, agene encoding each functional fragment is obtained according to themethod described above, and cells are transfected with the gene. Thefunctional fragment of interest can be collected from cultures of thecells.

The antibody of the present invention may be multimerized to therebyenhance its affinity for the antigen. The antibodies to be multimerizedmay be antibodies of one type or may be a plurality of antibodiesrecognizing a plurality of epitopes, respectively, or the same antigen.Examples of antibody multimerization methods can include the binding oftwo scFvs to an IgG CH3 domain, the binding thereof to streptavidin, andthe introduction of a helix-turn-helix motif.

The antibody of the present invention may be a mixture of plural typesof anti-ROBO4 antibodies differing in amino acid sequence, i.e., apolyclonal antibody. Examples of the polyclonal antibody can include amixture of plural types of antibodies differing in a portion or thewhole of the CDR set. Such a polyclonal antibody can be collected fromcultures of mixed-cultured different antibody-producing cells(WO2004/061104). Moreover, separately prepared antibodies may be mixed.Furthermore, antiserum, which is one aspect of the polyclonal antibody,can be prepared by immunizing animals with the desired antigen andcollecting serum from the animals according to a standard method.

Antibodies conjugated with various molecules such as polyethylene glycol(PEG) can also be used as modified forms of the antibody.

The antibody of the present invention may further be any of conjugatesformed by these antibodies with other drugs (immunoconjugates). Examplesof such an antibody can include the antibody conjugated with aradioactive material or a compound having a pharmacological effect(Nature Biotechnology (2005) 23, p. 1137-1146).

(4-7) Purification of Antibody

The obtained antibody can be purified into a homogeneous level. Usualprotein separation and purification methods can be used for theseparation and purification of the antibody.

The antibody can be separated and purified by appropriately selected orcombined approaches, for example, chromatography columns, filters,ultrafiltration, salting out, dialysis, preparative polyacrylamide gelelectrophoresis, and/or isoelectric focusing (Strategies for ProteinPurification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996);Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold SpringHarbor Laboratory (1988)) but not limited to them.

Examples of chromatography include affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtration,reverse-phase chromatography, and adsorption chromatography.

These chromatography approaches can be performed using liquid-phasechromatography such as HPLC or FPLC.

Examples of columns used in affinity chromatography can include proteinA, protein G, and antigen columns.

Examples of the protein A column include Hyper D (manufactured by PallCorp.), POROS (manufactured by Applied Biosystems, Inc.), and SepharoseF.F. (manufactured by GE Healthcare Bio-Sciences Corp.).

Also, the antibody may be purified based on its binding activity againstthe antigen using an antigen-immobilized carrier.

The present invention provides even a gene encoding the antibody of thepresent invention or the functional fragment thereof, or the modifiedform thereof, a recombinant vector containing an insert of the gene, acell transfected with the gene or the vector, and a cell producing theantibody of the present invention.

An antibody or functional fragment thereof which is produced by any ofthe methods (4-1) to (4-6) can be included in the present invention.

5. Pharmaceutical Composition

The present invention provides a pharmaceutical composition comprisingthe anti-ROBO4 antibody or the functional fragment thereof, or themodified form thereof.

The pharmaceutical composition of the present invention is useful in thetreatment or prevention of a disease that shows angiogenesis as one ofpathological findings during the course of onset, progression, and/orexacerbation and can be improved by the suppression of this angiogenesisor vascular permeability (hereinafter, this disease is referred to as an“angiogenic disease” for the sake of convenience). Examples of theangiogenic disease can include exudative age-related maculardegeneration, diabetic retinopathy, macular edema, benign or malignanttumor, atherosclerosis, retrolental fibroplasia, angioma, chronicinflammation, ocular neovascular disease, proliferative retinopathy,neovascular glaucoma, immune rejection of a corneal tissue transplant orother tissue transplants, rheumatoid arthritis, psoriasis, acuteinflammation, sepsis, and obesity. The pharmaceutical composition of thepresent invention is useful as an agent in the treatment or preventionof an angiogenic disease, preferably useful in the treatment orprevention of exudative age-related macular degeneration, diabeticretinopathy, macular edema, benign or malignant tumor, retrolentalfibroplasia, ocular neovascular disease, proliferative retinopathy,neovascular glaucoma, and immune rejection of a corneal tissuetransplant, more preferably useful in the treatment or prevention ofexudative age-related macular degeneration, diabetic retinopathy,macular edema, benign or malignant tumor, retrolental fibroplasia,ocular neovascular disease, proliferative retinopathy and neovascularglaucoma.

In the present invention, the treatment and/or treatment of a diseaseincludes, but not limited to, the prevention of onset of the disease,preferably the disease in an individual having the expressed ROBO4protein, the suppression or inhibition of exacerbation or progressionthereof, the alleviation of one or two or more symptoms exhibited by anindividual affected with the disease, the suppression or remission ofexacerbation or progression thereof, the treatment or prevention of asecondary disease, and the like.

The pharmaceutical composition of the present invention can contain atherapeutically or preventively effective amount of the anti-ROBO4antibody or the functional fragment of the antibody and apharmaceutically acceptable diluent, vehicle, solubilizer, emulsifier,preservative, and/or additive.

The “therapeutically or preventively effective amount” means an amountthat exerts therapeutic or preventive effects on a particular disease bymeans of a particular dosage form and administration route.

The pharmaceutical composition of the present invention may containmaterials for changing, maintaining, or retaining pH, osmotic pressure,viscosity, transparency, color, tonicity, sterility, or the stability,solubility, sustained release, absorbability, permeability, dosage form,strength, properties, shape, etc., of the composition or the antibodycontained therein (hereinafter, referred to as “pharmaceuticalmaterials”). The pharmaceutical materials are not particularly limitedas long as they are pharmacologically acceptable materials. For example,no or low toxicity is a property preferably possessed by thesepharmaceutical materials.

Examples of the pharmaceutical materials can include, but not limitedto, the followings: amino acids such as glycine, alanine, glutamine,asparagine, histidine, arginine, and lysine; antimicrobial agents;antioxidants such as ascorbic acid, sodium sulfate, and sodiumbisulfite; buffers such as phosphate, citrate, or borate buffers, sodiumbicarbonate, and Tris-HCl solutions; fillers such as mannitol andglycine; chelating agents such as ethylenediaminetetraacetic acid(EDTA); complexing agents such as caffeine, polyvinylpyrrolidine,β-cyclodextrin, and hydroxypropyl-β-cyclodextrin; bulking agents such asglucose, mannose, and dextrin; other hydrocarbons such asmonosaccharides, disaccharides, glucose, mannose, and dextrin; coloringagents; corrigents; diluents; emulsifiers; hydrophilic polymers such aspolyvinylpyrrolidine; low-molecular-weight polypeptides; salt-formingcounterions; antiseptics such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, and hydrogen peroxide;solvents such as glycerin, propylene glycol, and polyethylene glycol;sugar alcohols such as mannitol and sorbitol; suspending agents;surfactants such as PEG, sorbitan ester, polysorbates such aspolysorbate 20 and polysorbate 80, triton, tromethamine, lecithin, andcholesterol; stability enhancers such as sucrose and sorbitol;elasticity enhancers such as sodium chloride, potassium chloride,mannitol, and sorbitol; transport agents; diluents; excipients; and/orpharmaceutical additives.

The amount of these pharmaceutical materials added is 0.001 to 1000times, preferably 0.01 to 100 times, more preferably 0.1 to 10 times theweight of the anti-ROBO4 antibody or the functional fragment thereof, orthe modified form thereof.

A pharmaceutical composition containing an immunoliposome comprising theanti-ROBO4 antibody or the functional fragment thereof, or the modifiedform thereof encapsulated in a liposome or a modified antibody formcomprising the antibody conjugated with a liposome (U.S. Pat. No.6,214,388, etc.) is also included in the pharmaceutical composition ofthe present invention.

The excipients or vehicles are not particularly limited as long as theyare liquid or solid materials usually used in injectable water, saline,artificial cerebrospinal fluids, and other preparations for oral orparenteral administration. Examples of the saline can include neutralsaline and serum albumin-containing saline.

Examples of the buffers can include Tris buffers adjusted to bring aboutthe final pH of the pharmaceutical composition to 7.0 to 8.5, acetatebuffers adjusted to bring about the final pH thereof to 4.0 to 5.5,citrate buffers adjusted to bring about the final pH thereof to 5.0 to8.0, and histidine buffers adjusted to bring about the final pH thereofto 5.0 to 8.0.

The pharmaceutical composition of the present invention is a solid, aliquid, a suspension, or the like. Another example of the pharmaceuticalcomposition of the present invention can include freeze-driedpreparations. The freeze-dried preparations can be formed using anexcipient such as sucrose.

The administration route of the pharmaceutical composition of thepresent invention may be any of enteral administration, localadministration, and parenteral administration and may be selectedpreferably according to the targeted disease. Specific examples thereofcan include intravenous administration, intraarterial administration,intramuscular administration, intradermal administration, hypodermicadministration, intraperitoneal administration, transdermaladministration, intraosseous administration, and intraarticularadministration. Also, intraocular administration can be used preferablyfor an ophthalmic angiogenic disease such as exudative age-relatedmacular degeneration, diabetic retinopathy, macular edema, retrolentalfibroplasia, ocular neovascular disease, proliferative retinopathy,neovascular glaucoma, or immune rejection of a corneal tissuetransplant.

The recipe of the pharmaceutical composition can be determined accordingto the administration method, the binding affinity of the antibody forthe ROBO4 protein, etc. The anti-ROBO4 antibody of the present inventionor the functional fragment thereof, or the modified form thereof havinghigher affinity (lower K_(D) value) for the ROBO4 protein can exert itsdrug efficacy at a lower dose.

The dose of the anti-ROBO4 antibody of the present invention can bedetermined appropriately according to the species of an individual, thetype of a disease, symptoms, sex, age, pre-existing conditions, thebinding affinity of the antibody for the ROBO4 protein or its biologicalactivity, and other factors. A dose of usually 0.01 to 1000 mg/kg,preferably 0.1 to 100 mg/kg, can be administered once every day to 180days or twice or three or more times a day.

Examples of the form of the pharmaceutical composition can includeinjections (including freeze-dried preparations and drops),suppositories, transnasal absorption preparations, transdermalabsorption preparations, sublingual formulations, capsules, tablets,ointments, granules, aerosols, pills, powders, suspensions, emulsions,eye drops, and biological implant formulations.

The pharmaceutical composition comprising the anti-ROBO4 antibody of thepresent invention or the functional fragment thereof, or the modifiedform thereof as an active ingredient may be used in combination with afurther therapeutic or prophylactic agent. Examples of said agentinclude an anti-angiogenesis drug, anti-inflammatory drug, and/or ananticancer drug. For example, the anti-angiogenesis drug,anti-inflammatory drug, and/or anticancer drug is administered to asubject, and then, the pharmaceutical composition comprising theanti-ROBO4 antibody or the functional fragment of the antibody as anactive ingredient is administered thereto. Alternatively, thepharmaceutical composition is administered to a subject, and then, theanti-angiogenesis drug, anti-inflammatory drug, and/or anticancer drugis administered thereto. Alternatively, the pharmaceutical compositionmay be administered to a subject simultaneously with theanti-angiogenesis drug, anti-inflammatory drug, and/or anticancer drug.Examples of the anti-angiogenesis drug can include ranibizumab.

The present invention provides even a method for treating or preventingan angiogenic disease such as exudative age-related maculardegeneration, diabetic retinopathy, macular edema, benign or malignanttumor, atherosclerosis, retrolental fibroplasia, angioma, chronicinflammation, ocular neovascular disease, proliferative retinopathy,neovascular glaucoma, immune rejection of a corneal tissue transplant orother tissue transplants, rheumatoid arthritis, psoriasis, acuteinflammation, sepsis, or obesity, use of the antibody of the presentinvention for the preparation of a pharmaceutical composition for thetreatment or prevention of the angiogenic disease, and use of theantibody of the present invention for the treatment or prevention of theangiogenic disease. A kit for treatment or prevention comprising theantibody of the present invention is also included in the presentinvention.

6. Composition for Diagnosis

The present invention provides a composition for examination ordiagnosis comprising the anti-ROBO4 antibody of the present invention orthe functional fragment thereof, or the modified form thereof(hereinafter, collectively referred to as a “composition fordiagnosis”).

The composition for diagnosis of the present invention is useful in theexamination or diagnosis of an angiogenic disease such as exudativeage-related macular degeneration, diabetic retinopathy, macular edema,benign or malignant tumor, atherosclerosis, retrolental fibroplasia,angioma, chronic inflammation, ocular neovascular disease, proliferativeretinopathy, neovascular glaucoma, immune rejection of a corneal tissuetransplant or other tissue transplants, rheumatoid arthritis, psoriasis,acute inflammation, sepsis, or obesity. The composition for diagnosis ofthe present invention is also useful in the examination or diagnosis ofearly angiogenesis or pre-angiogenesis symptoms, which do not satisfythe conventional diagnosis criteria, undiagnosed symptoms that evolve toangiogenesis, etc. In the present invention, the examination or thediagnosis includes, for example, the determination or testing of a riskof acquiring a disease, the determination of the presence or absence ofa disease, the testing of the degree of progression or exacerbation, thetesting or determination of the effect of drug therapy using thepharmaceutical composition comprising the anti-ROBO4 antibody or thelike, the testing or determination of the effect of therapy other thandrug therapy, the testing of a risk of recurrence, and the determinationof the presence or absence of recurrence. However, the examination orthe diagnosis according to the present invention is not limited to themas long as it is usual examination or diagnosis.

When the ROBO4 protein is detected in a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20-fold or more amount, preferably10-fold or more amount, in a sample derived from a test subject comparedwith a sample derived from a healthy individual, the test subject can bediagnosed as having an angiogenic disease or as being at a high risk ofacquiring it. Moreover, when the serum concentration of the ROBO4protein exceeds a particular reference value, the test subject isdiagnosed as having an angiogenic disease or can be diagnosed as beingat a high risk of acquiring it. The reference value is usually 0.01 to10 ng/ml, preferably 0.1 to 1 ng/ml, more preferably 0.1 to 0.3 ng/ml.

Such a composition for diagnosis can contain a pH buffer, anosmoregulator, salts, a stabilizer, an antiseptic, a color developer, asensitizer, an aggregation inhibitor, and the like.

The present invention provides even a method for examining or diagnosingan angiogenic disease such as exudative age-related maculardegeneration, diabetic retinopathy, macular edema, benign or malignanttumor, atherosclerosis, retrolental fibroplasia, angioma, chronicinflammation, ocular neovascular disease, proliferative retinopathy,neovascular glaucoma, immune rejection of a corneal tissue transplant orother tissue transplants, rheumatoid arthritis, psoriasis, acuteinflammation, sepsis, or obesity, use of the antibody of the presentinvention for the preparation of a composition for the diagnosis of theangiogenic disease, and use of the antibody of the present invention forthe examination or diagnosis of the angiogenic disease. A kit forexamination or diagnosis comprising the antibody of the presentinvention is also included in the present invention.

The examination or diagnosis method involving the antibody of thepresent invention is preferably sandwich ELISA. A usual detection methodusing antibodies, such as ELISA, RIA, ELISPOT (enzyme-linked immunospot)assay, dot blotting, an Ouchterlony test, or CIE(counterimmunoelectrophoresis), may be used. Antibodies applied to thesandwich ELISA assay system may be any combination of two antibodiesthat recognize ROBO4, but do not compete with each other. In addition tobiotin, a labeling method that can be carried out in biochemicalanalysis, such as HRP, alkaline phosphatase, or FITC, can be used as alabeling method for the antibodies. A chromogenic substrate such as TMB(3,3′,5,5′-tetramethylbenzidine), BCIP (5-bromo-4-chloro-3-indolylphosphate), ρ-NPP (ρ-nitrophenyl phosphate), OPD (o-Phenylenediamine),ABTS (3-Ethylbenzothiazoline-6-sulfonic acid), SuperSignal ELISA PicoChemiluminescent Substrate (Thermo Fisher Scientific Inc.), afluorescent substrate such as QuantaBlu™ Fluorogenic PeroxidaseSubstrate (Thermo Fisher Scientific Inc.), and a chemiluminescentsubstrate can be used in detection using enzymatic labeling. Samplesderived from human or non-human animals as well as artificially treatedsamples such as recombinant proteins can be subjected to this assay.Examples of test samples derived from organism individuals can include,but not limited to, blood, synovial fluids, ascites, lymph,cerebrospinal fluids, and tissue homogenate supernatants.

The sandwich ELISA kit for examination or diagnosis comprising theantibody of the present invention may contain a solution of ROBO4protein standards, a coloring reagent, a buffer solution for dilution,an antibody for solid phase, antibody for detection, and a washingsolution, and the like. The amount of the antibody bound to the antigencan be measured preferably using a method such as an absorbance,fluorescence, luminescence, or RI (radioisotope) method. An absorbanceplate reader, a fluorescence plate reader, a luminescence plate reader,an RI liquid scintillation counter, or the like is preferably used inthe measurement.

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to Examples. However, the present invention is not intended tobe limited to them.

In the Examples below, each operation for genetic engineering wasperformed by methods described in “Molecular Cloning” (Sambrook, J.,Fritsch, E. F., and Maniatis, T., Cold Spring Harbor Laboratory Press,1989) or methods described in other experimental manuals used by thoseskilled in the art, or performed according to the instructions ofcommercially available reagents or kits used, unless otherwisespecified.

(Example 1) Preparation of Expression Vector 1)-1 Preparation of HumanROBO4 Expression Vector

1)-1-1 Preparation of Full-Length Human ROBO4 Expression Vector

Human ROBO4 cDNA was cleaved off with EcoRV and NotI from a plasmid(manufactured by Open Biosystems) comprising human ROBO4 cDNA (AccessionNo. BC039602) and incorporated between EcoRV and NotI of a pCI vector(manufactured by Promega Corp.) to prepare a full-length human ROBO4expression vector (hereinafter, referred to as “pCI-hROBO4”). Thesequence of the human ROBO4 gene cloned in this vector is shown in SEQID NO: 1. Also, the amino acid sequence of human ROBO4 is shown in SEQID NO: 2.

1)-1-2 Preparation of Human ROBO4 Extracellular Region Expression Vector

cDNA encoding a human ROBO4 extracellular region polypeptide (consistingof an amino acid sequence represented by amino acid Nos. 1 to 461 of SEQID NO: 2; hereinafter, abbreviated to “human ROBO4-ECD”) was amplifiedthrough PCR reaction using a primer set:

primer 1F: (SEQ ID NO: 73) 5′-aaaggtaccaccatgggctctggaggagacagcctcctg-3′and primer 1R: (SEQ ID NO: 74)5′-aaagatatcctgctccagggtccagggaccatgctcact-3′.The obtained PCR product was cloned into a pEF6/V5-His-TOPO vector(manufactured by Life Technologies Corp.) (hereinafter, the resultingvector is abbreviated to “pEF6-ROBO4-ECD”; hereinafter, a recombinantprotein expressed by “pEF6-ROBO4-ECD” is referred to as “rROBO4-ECD”).1)-1-3 Preparation of N-Terminal FLAG-Tagged Full-Length Human ROBO4 andHuman ROBO4 Extracellular Region/Domain Deletion Variant ExpressionVectors

In order to construct vectors for expression of a protein comprising aregion consisting of an amino acid sequence represented by amino acidNos. 28 to 1007 of SEQ ID NO: 2 of human ROBO4 (in the diagram, thisregion is referred to as “hROBO4-28”), a region consisting of an aminoacid sequence represented by amino acid Nos. 46 to 1007 thereof (in thediagram, this region is referred to as “hROBO4-46”), a region consistingof an amino acid sequence represented by amino acid Nos. 132 to 1007thereof (in the diagram, this region is referred to as “hROBO4-132”), aregion consisting of an amino acid sequence represented by amino acidNos. 210 to 1007 thereof (in the diagram, this region is referred to as“hROBO4-210”), a region consisting of an amino acid sequence representedby amino acid Nos. 225 to 1007 thereof (in the diagram, this region isreferred to as “hROBO4-225”), or a region consisting of an amino acidsequence represented by amino acid Nos. 341 to 1007 thereof (in thediagram, this region is referred to as “hROBO4-341”) with the N-terminustagged with FLAG, PCR reaction was performed with pCI-hROBO4 as atemplate using each primer set:

primer set for hROBO4-28 amplification: primer 2F: (SEQ ID NO: 75)5′-ggtaccgccatgggctctggaggagacagcctcctcggcggcagaggttccctgcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataagcaggactccccgccccagatcctagtc cac-3′ and primer 2R:(SEQ ID NO: 76) 5′-gctagcggagtaatctacaggagaagcaccagccttg-3′,primer set for hROBO4-46 amplification: primer 3F: (SEQ ID NO: 77)5′-ggtaccgccatgggctctggaggagacagcctcctcggcggcagaggttccctgcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataagcctggccctgccaggatgagctgccaa  g-3′ and the primer 2Rprimer set for hROB04-132 amplification: primer 4F: (SEQ ID NO: 78)5′-ggtaccgccatgggctctggaggagacagcctcctcggcggcagaggttccctgcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataaggtggctgtcctccgggaggatttccag atc-3′and the primer 2R, primer set for hROB04-210 amplification: primer 5F:(SEQ ID NO: 79) 5′-ggtaccgccatgggctctggaggagacagcctcctcggcggcagaggttccctgcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataagaccaacagcgcaggacatagggagagc c-3′ and the primer 2R,primer set for hROBO4-225 amplification: primer 6F: (SEQ ID NO: 80)5′-ggtaccgccatgggactggaggagacagcctcctcggcggcagaggttccctgcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataagatccaggagccccaggactacacggagc c-3′ and the primer 2R,primer set for hROBO4-341 amplification: primer 7F: (SEQ ID NO: 81)5′-ggtaccgccatgggctaggaggagacagcctcctcggcggcagaggttccctgcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataagaggctgccggaaaaagtgcccagtgccc ca-3′and the primer 2R.and the primer 2R. The obtained PCR product was incorporated into apCR4Blunt-TOPO vector (manufactured by Life Technologies Corp.) toprepare a cloning vector. From each cloning vector, the correspondingcDNA was cleaved off with KpnI and NheI and incorporated between KpnIand NheI of a pCI vector to prepare an N-terminal FLAG-taggedfull-length human ROBO4 expression vector and four human ROBO4extracellular region/domain deletion variant expression vectors. TheN-terminal FLAG-tagged full-length human ROBO4 vector consists of anucleotide encoding the signal sequence of ROBO4 (amino acids of aminoacid Nos. 1 to 27 of SEQ ID NO: 2)+FLAG sequence (DYKDDDDK)+ROBO4 (aminoacids of amino acid Nos. 28 to 1007 of SEQ ID NO: 2) from theN-terminus. Hereinafter, the vector for expression of N-terminalFLAG-tagged full-length human ROBO4 is referred to as“pCI-FLAG-hROBO4-28”. Of the human ROBO4 extracellular region/domaindeletion variant expression vectors, for example, the vector forexpression of ROBO4 consisting of amino acid Nos. 46 to 1007 of SEQ IDNO: 2 consists of a nucleotide encoding the signal sequence of ROBO4(amino acids of amino acid Nos. 1 to 27 of SEQ ID NO: 2)+FLAG sequence(DYKDDDDK)+extracellular region-deleted ROBO4 (amino acids of amino acidNos. 46 to 1007 of SEQ ID NO: 2). This vector is referred to as“pCI-hROBO4-46”. Likewise, the vectors encoding ROBO4 having a partialdeletion in the extracellular region of ROBO4 are referred to as“pCI-hROBO4-132”, “pCI-hROBO4-210”, “pCI-hROBO4-225”, and“pCI-hROBO4-341”, respectively.

The nucleotide sequence encoding the amino acid sequence of FLAG-taggedfull-length human ROBO4 or each extracellular region/domain deletionvariant of human ROBO4 cloned in the vector is shown in SEQ ID NO: 3, 5,7, 9, 11, or 13. Also, the amino acid sequence of the correspondingFLAG-tagged full-length human ROBO4 or extracellular region/domaindeletion variant of human ROBO4 is shown in SEQ ID NO: 4, 6, 8, 10, 12,or 14.

1)-1-4 Preparation of Human ROBO4 Intracellular Region Deletion VariantExpression Vector

In order to construct a vector for expression of a protein comprising aregion consisting of an amino acid sequence represented by amino acidNos. 1 to 511 of SEQ ID NO: 2 of human ROBO4 (hereinafter, this regionis referred to as “hROBO4-ΔC”), a stop codon was inserted immediatelyafter a codon encoding the 511th amino acid of human ROBO4 withpCI-hROBO4 as a template using primer set for hROBO4-ΔC:

primer 8F: (SEQ ID NO: 82)5′-cagatataccagtgaggatgcctgaatcctaaaacacaggatggat c-3′ and primer 8R:(SEQ ID NO: 83) 5′-gatccatcctgtgttttaggattcaggcatcctcactggtatatct g-3′,and QuikChange XL Site-Directed Mutagenesis Kit (manufactured by AgilentTechnologies, Inc.) to prepare a hROBO4-ΔC expression vector(hereinafter, referred to as “pCI-hROBO4-ΔC”).

1)-2 Preparation of Mouse ROBO4 Expression Vector

PCR reaction was performed with Mouse Heart QUICK-Clone cDNA(manufactured by Takara Bio Inc.) as a template using a primer set:

primer 9F: (SEQ ID NO: 84)5′-ggtaccgccatgggacaaggagaggagccgagagcagccatg-3′ and primer 9R:(SEQ ID NO: 85) 5′-gcggccgcggaggaatcaccagecttgggcacagcaccag-3′.The obtained PCR product was incorporated into a pCR-Blunt II-TOPOvector (manufactured by Life Technologies Corp.) to prepare a cloningvector comprising mouse ROBO4 cDNA. From the cloning vector, mouse ROBO4cDNA was cleaved off with KpnI and NotI and incorporated between KpnIand NotI of a pCI vector to prepare a mouse ROBO4 expression vector(hereinafter, referred to as “pCI-mROBO4”). The sequence of an ORF sitein the mouse ROBO4 gene cloned in this vector is shown in nucleotideNos. 7 to 3051 of SEQ ID NO: 15. Also, the amino acid sequence of mouseROBO4 is shown in SEQ ID NO: 16.

1)-3 Preparation of Rat ROBO4 Expression Vector

PCR reaction was performed with Rat Spleen QUICK-Clone cDNA(manufactured by Takara Bio Inc.) as a template using a primer set:

primer 10F: (SEQ ID NO: 86)5′-ggtaccgccatgggacaaggagaggagctgagagcagcc-3′ and primer 10R:(SEQ ID NO: 87) 5′-gcggccgcggaggaatcaccagccttgggcacaacacc-3′.The obtained PCR product was incorporated into a pCR4Blunt-TOPO vectorto prepare a cloning vector comprising rat ROBO4 cDNA. From the cloningvector, rat ROBO4 cDNA was cleaved off with KpnI and NotI andincorporated between KpnI and NotI of a pCI vector to prepare a ratROBO4 expression vector (hereinafter, referred to as “pCI-raROBO4”). Thenucleotide sequence of rat ROBO4 cDNA is shown in SEQ ID NO: 17. Theamino acid sequence encoded by this nucleotide sequence is shown in SEQID NO: 18.

1)-4 Preparation of N-Terminal FLAG-Tagged Cynomolgus Monkey ROBO4Expression Vector

PCR reaction was performed with cDNA synthesized from cynomolgus monkeykidney total RNA as a template using a primer set:

primer 11F: (SEQ ID NO: 88) 5′-ggtaccgccatgggctctggaggagaaagcctccggg-3′and primer 11R: (SEQ ID NO: 89) 5′-ggagtaatctacaggagaagcaccagccttg-3′.The obtained PCR product was incorporated into a pCR4Blunt-TOPO vectorto prepare two types of cloning vectors comprising each cynomolgusmonkey ROBO4 cDNA (hereinafter, referred to as cynoROBO4-1 orcynoROBO4-2) (hereinafter, these vectors are referred to aspCR-cynoROBO4-1 and pCR-cynoROBO4-2, respectively).

Next, PCR reaction was performed with pCR-cynoROBO4-1 or pCR-cynoROBO4-2as a template using a primer set:

primer 12F: (SEQ ID NO: 90) 5′-ggatccgccatgggctctggaggagaaagcctccg-3′and primer 12R: (SEQ ID NO: 91)5′-gcggccgctcaggagtaatctacaggagaagcaccagccttg-3′.The obtained PCR product was incorporated into a pCR4Blunt-TOPO vectorto prepare a cloning vector comprising each cynomolgus monkey ROBO4cDNA. From the cloning vector, the corresponding cynomolgus monkey ROBO4cDNA was cleaved off with BamHI and NotI and incorporated between BamHIand NotI of a pCI vector to prepare two types of cynomolgus monkey ROBO4expression vectors (hereinafter, referred to as pCI-cynoROBO4-1 andpCI-cynoROBO4-2, respectively).

Next, PCR reaction was performed with pCI-cynoROBO4-1 or pCI-cynoROBO4-2as a template using a primer set:

primer 13F: (SEQ ID NO: 92)5′-ggtaccgccatgggctctggaggagaaagcctccgaggctcccgggcttcccggcctctgctgctcctgctcatcatgggaggcatggctgattacaaggatgacgacgataagcaggactccccgccccagatcctagt ccac-3′and the primer 12R.The obtained PCR product was incorporated into a pCR-TOPO vector(manufactured by Life Technologies Corp.) to prepare each cloning vectorcomprising N-terminal FLAG-tagged cynomolgus monkey ROBO4 cDNA. From thecloning vector, the corresponding cynomolgus monkey ROBO4 cDNA wascleaved off with KpnI and NotI and incorporated between KpnI and NotI ofa pCI vector to prepare N-terminal FLAG-tagged cynomolgus monkey ROBO4expression vectors (hereinafter, referred to as “pCI-FLAG-cynoROBO4-1”and “pCI-FLAG-cynoROBO4-2”, respectively). The nucleotide sequence ofcynomolgus monkey ROBO4 cDNA cloned in each of pCI-FLAG-cynoROBO4-1 andpCI-FLAG-cynoROBO4-2 is shown in SEQ ID NOs: 19 and 21, respectively.The amino acid sequence encoded by each nucleotide sequence is shown inSEQ ID NOs: 20 and 22, respectively.

1)-5 Preparation of N-Terminal FLAG-Tagged Human ROBO1 Expression Vector

PCR reaction was performed with Human Heart QUICK-Clone cDNA(manufactured by Takara Bio Inc.) as a template using a primer set:

primer 13F: (SEQ ID NO: 93)5′-ggggacaagtttgtacaaaaaagcaggcttcaccatgattgcgg agcccgctcacttttacctg-3′and primer 13R: (SEQ ID NO: 94)5′-ggggaccactttgtacaagaaagctgggtcgctttcagtttcct ctaattcttc-3′.The obtained PCR product and a pDONR221 vector (manufactured by LifeTechnologies Corp.) were subjected to BP reaction to prepare a donorvector comprising human ROBO1 cDNA.

Next, the PCR reaction was performed with the donor vector as a templateusing a primer set:

primer 14F: (SEQ ID NO: 95)5′-gcggccgcatgattgcggagcccgctcacttttacctgtttggattaatatgtctctgttcaggctcccgtcttgattacaaggatgacgacgataagcgtcaggaagattttccacctcgcattgttg-3′ and primer 14R:(SEQ ID NO: 96) 5′-gctagctcagctttcagtttcctctaattcttc-3′.The obtained PCR product was incorporated into a pCR4Blunt-TOPO vectorto prepare a cloning vector comprising N-terminal FLAG-tagged humanROBO1 cDNA. From the cloning vector, N-terminal FLAG-tagged human ROBO1cDNA was cleaved off with NheI and NotI and incorporated between NheIand NotI of a pCI vector to prepare an expression vector (hereinafter,referred to as “pCI-FLAG-hROBO1”). The nucleotide sequence of N-terminalFLAG-tagged human ROBO1 cDNA is shown in SEQ ID NO: 23. The amino acidsequence encoded by this nucleotide sequence is shown in SEQ ID NO: 24.

1)-6 Preparation of Human ROBO2 Expression Vector

PCR reaction was performed with Human Lung QUICK-Clone cDNA(manufactured by Takara Bio Inc.) as a template using a primer set:

primer 15F: (SEQ ID NO: 97) 5′-gcggccgcatgagtctgctgatgtttacacaactactg-3′and primer 15R: (SEQ ID NO: 98)5′-gctagcctataattcacctgtaaactgtccttgactgttg-3′.The obtained PCR product was incorporated to a pCR4Blunt-TOPO vector toprepare a cloning vector comprising human ROBO2 cDNA. From the cloningvector, human ROBO2 cDNA was cleaved off with NotI and NheI andincorporated between NotI and NheI of a pCI vector to prepare anexpression vector (hereinafter, referred to as “pCI-hROBO2”). Thenucleotide sequence of human ROBO2 cDNA is shown in SEQ ID NO: 25. Theamino acid sequence encoded by this nucleotide sequence is shown in SEQID NO: 26.

1)-7 Preparation of Human ROBO3 Expression Vector

PCR reaction was performed with human ROBO3/pENTR223.1 (manufactured byOpen Biosystems) as a template using a primer set:

primer 16F: (SEQ ID NO: 99) 5′-gcggccgcatgctgcgctacctgctgaaaacgctgctg-3′and primer 16R: (SEQ ID NO: 100)5′-gctagctcatcttggttcctctcggcgtttctgtcc-3′.The obtained PCR product was incorporated to a pCR4Blunt-TOPO vector toprepare a cloning vector comprising human ROBO3 cDNA. From the cloningvector, human ROBO3 cDNA was cleaved off with NotI and NheI andincorporated between NotI and NheI of a pCI vector to prepare anexpression vector (hereinafter, referred to as “pCI-hROBO3”). Thesequence of an ORF site in the human ROBO3 gene cloned in this vector isshown in nucleotide Nos. 35 to 4192 of SEQ ID NO: 27. Also, the aminoacid sequence of human ROBO3 is shown in SEQ ID NO: 28.

(Example 2) Preparation of Monoclonal Antibody 2)-1 Preparation ofAntigenic Protein

In order to express rROBO4-ECD, FreeStyle 293-F cells (manufactured byLife Technologies Corp.) were transfected with pEF6-ROBO4-ECD using293fectin (manufactured by Life Technologies Corp.) and cultured at 37°C. for 6 days under 8% CO₂ conditions. After completion of the culture,the culture solution was collected by centrifugation and used as arROBO4-ECD purification stock. The obtained culture supernatant wasdialyzed against 20 mM Tris-HCl, pH 7.5 using a dialysis tube having amolecular weight cutoff of 15000, filtered through a filter (0.45 μm),and then added to HiTrap 16/10 Q XL (manufactured by GE HealthcareBio-Sciences Corp.) equilibrated with 20 mM Tris-HCl, pH 7.5. Elutionwas performed with a NaCl gradient (20 mM Tris-HCl, pH 7.5/0.2 M NaCl,20 mM Tris-HCl, pH 7.5/1 M NaCl). A portion of the elution fraction wasseparated by SDS-polyacrylamide gel electrophoresis (hereinafter,abbreviated to “SDS-PAGE”). Then, the gel was subjected to CoomassieBrilliant Blue staining (hereinafter, abbreviated to “CBB staining”) anddetection by Western blotting to confirm a fraction containingrROBO4-ECD. Next, the fraction containing rROBO4-ECD was collected andadded to HiLoad 16/60 Superdex 75 pg (manufactured by GE HealthcareBio-Sciences Corp.) equilibrated with PBS. After elution with PBS, aportion of the elution fraction was separated by SDS-PAGE. Then, the gelwas subjected to CBB staining and detection by Western blotting toconfirm a fraction containing rROBO4-ECD. The fraction containingrROBO4-ECD was collected and used as an antigen for immunization and asan antigen for binding affinity assay. The protein concentration wasmeasured using BCA Protein Assay Reagent (manufactured by PierceBiotechnology, Inc.).

2)-2 Immunization

Six-week-old female BALB/c mice were used. At day 0, 50 μg of a mixtureof rROBO4-ECD and a Freund's complete adjuvant was hypodermically orintradermally administered to each mouse. At days 7, 14, and 21, 50 μgof a mixture of rROBO4-ECD and a Freund's incomplete adjuvant washypodermically or intradermally administered to the mouse. At day 38, 50μg of rROBO4-ECD was intraperitoneally administered to the mouse. At day42, the mouse lymph node or spleen was collected and used in hybridomapreparation.

2)-3 Hybridoma Preparation

The lymph node cells or spleen cells and mouse myeloma SP2/0-ag14 cellswere electrically fused using Hybrimune Hybridoma Production System(manufactured by Cyto Pulse Sciences, Inc.), diluted with ClonaCell-HYSelection Medium D (manufactured by StemCell Technologies Inc.), andcultured. Hybridoma colonies that appeared were collected to preparemonoclonal hybridomas. Each hybridoma colony collected was cultured, andthe obtained hybridoma culture supernatant was screened for anti-ROBO4antibody-producing hybridomas.

2)-4 Antibody Screening

2)-4-1 Preparation of Antigen Gene-Expressing Cell for Cell-ELISA

HEK293 cells were adjusted to 7.5×10⁵ cells/mL in a DMEM mediumcontaining 10% FBS. The cells were transfected with pCI-hROBO4 or anegative control pCI-mock using Lipofectamine 2000 (manufactured by LifeTechnologies Corp.), dispensed at a concentration of 50 μL/well to a96-well half area plate (manufactured by Corning Inc.), and culturedovernight in a DMEM medium containing 10% FBS at 37° C. under 5% CO₂conditions. The obtained transfected cells were used in Cell-ELISA withthem adhering to each other.

2)-4-2 Preparation of Antigen Gene-Expressing Cell for Flow CytometryAnalysis

HEK293T cells were inoculated at a concentration of 1.125×10⁷cells/flask to a 225-cm² flask (manufactured by Sumitomo Bakelite Co.,Ltd.) and cultured overnight in a DMEM medium containing 10% FBS at 37°C. under 5% CO₂ conditions. On the next day, the HEK293T cells weretransfected with pCI-ROBO4 or a negative control pCI-mock usingLipofectamine 2000 and further cultured overnight at 37° C. under 5% CO₂conditions. On the next day, the expression vector-transfected HEK293Tcells were treated with TrypLE Express (manufactured by LifeTechnologies Corp.), washed with DMEM containing 10% FBS, and thensuspended in PBS containing 5% FBS. The obtained cell suspension wasused in flow cytometry analysis.

2)-5 Cell-ELISA

After removal of a supernatant from the expression vector-transfectedHEK293 cells prepared in 2)-4-1, the hybridoma culture supernatant wasadded to each of the pCI-hROBO4- and pCI-mock-transfected HEK293 cells,and the cells were left standing at 4° C. for 1 hour. The cells in eachwell were washed once with PBS containing 5% FBS. Then, Anti-MouseIgG-Peroxidase antibody produced in goat (manufactured by Sigma-AldrichCorp.) diluted 500-fold with PBS containing 5% FBS was added thereto,and the cells were left standing at 4° C. for 1 hour. The cells in eachwell were washed 5 times with PBS containing 5% FBS. Then, an OPDcoloring solution (o-phenylenediamine dihydrochloride (manufactured byWako Pure Chemical Industries, Ltd.) and H₂O₂ dissolved atconcentrations of 0.4 mg/mL and 0.6% (v/v), respectively, in an OPDdissolving solution (0.05 M trisodium citrate, 0.1 M disodium hydrogenphosphate dodecahydrate, pH 4.5)) was added thereto at a concentrationof 25 μL/well. Color reaction was performed with intermittent stirringand stopped by addition of 1 M HCl at a concentration of 25 μL/well.Then, absorbance at 490 nm was measured using a plate reader (ENVISION;manufactured by Perkin Elmer, Inc.). In order to select hybridomasproducing an antibody specifically binding to ROBO4 expressed on cellmembrane surface, hybridomas in the culture supernatants exhibitinghigher absorbance in the pCI-hROBO4-transfected HEK293 cells comparedwith the negative control pCI-mock-transfected HEK293 cells wereselected as anti-ROBO4 antibody production-positive hybridomas.

2)-6 Flow Cytometry Analysis

The antibody produced by each hybridoma determined to be positive in2)-5 Cell-ELISA was further confirmed to bind to ROBO4 by flowcytometry. The HEK293T cell suspension prepared in 2)-4-2 wascentrifuged. After removal of the supernatant, the hybridoma culturesupernatant was added to each of the pCI-hROBO4-transfected cells andthe pCI-mock-transfected cells, and the resulting suspension was leftstanding at 4° C. for 1 hour. The cells were washed twice with PBScontaining 5% FBS. Then, Anti-Mouse IgG FITC conjugate (manufactured bySigma-Aldrich Corp.) diluted 1000-fold with PBS containing 5% FBS orAnti-Rat IgG FITC conjugate (manufactured by Sigma-Aldrich Corp.)diluted 320-fold with PBS containing 5% FBS was added thereto, and theresulting suspension was left standing at 4° C. for 1 hour. The cellswere washed three times with PBS containing 5% FBS, then resuspended inPBS containing 5% FBS and 2 μg/mL 7-aminoactinomycin D (manufactured byMolecular Probes), and subjected to detection using a flow cytometer(FC500; manufactured by Beckman Coulter, Inc.). Data was analyzed usingFlowjo (manufactured by TreeStar Inc.). 7-aminoactinomycin D-positivedead cells were excluded by gating, and the histogram of FITCfluorescence intensity was then prepared for live cells. Hybridomasproducing a sample in which the histogram of the pCI-ROBO4-transfectedHEK293T cells was shifted to a stronger fluorescence intensity regioncompared with the histogram of the negative control pCI-mock-transfected293T cells were obtained as anti-ROBO4 antibody-producing hybridomas.Anti-ROBO4 antibodies produced by the obtained hybridomas weredesignated as MAb1, MAb2, MAb3, and MAb4, respectively.

2)-7 Isotyping of Monoclonal Antibody

The isotype of each monoclonal antibody was determined using Mousemonoclonal isotyping kit or Rat monoclonal isotyping kit (manufacturedby AbD Serotec). The results were IgG1 (MAb1 and MAb2) and IgG2b (MAb3and MAb4).

2)-8 Preparation of Monoclonal Antibody

Each monoclonal antibody was purified from a hybridoma culturesupernatant (hereinafter, referred to as an “antibody purificationstock”).

The antibody purification stock was prepared as follows: 8 to 9×10⁷hybridomas were inoculated to a 1272-cm² flask (manufactured by CorningInc.) and cultured in a hybridoma SFM medium (manufactured by LifeTechnologies Corp.) containing 20% Ultra-LoW IgG fetal bovine serum at37° C. for 4 days under 5% CO₂ conditions, and the supernatant was thencollected.

The antibody was purified using Hitrap Protein G HP or Hitrap Mab SelectSuRe (manufactured by GE Healthcare Bio-Sciences Corp.). For HitrapProtein G HP, the antibody purification stock was added to a column andwashed with a binding buffer (0.02 M sodium phosphate, pH 7.0), followedby elution with 0.1 M glycine, pH 2.7. By contrast, for Hitrap MabSelect SuRe, the antibody purification stock was added to a column andwashed with PBS, followed by elution with 2 M arginine-HCl, pH 4.0. Theeluted antibody solution was neutralized, and the buffer was thenreplaced by PBS. The concentration of the antibody purified with HitrapProtein G HP was measured using BCA Protein Assay Reagent. Mouse IgG2a(manufactured by R&D systems, Inc.) was used as a standard for acalibration curve. Alternatively, the concentration of the antibodypurified with Hitrap Mab Select SuRe was determined by the measurementof absorbance (O.D. 280 nm) in an eluate of the antibody bound to POROSG 20 μm Column PEEK, 4.6 mm×50 mm, 0.83 mL (manufactured by AppliedBiosystems, Inc.). Specifically, the antibody sample diluted with PBSwas added to POROS G 20 μm equilibrated with an equilibration buffer(30.6 mM sodium dihydrogen phosphate dodecahydrate, 19.5 mMmonopotassium phosphate, 0.15 M NaCl, pH 7.0). The column was washedwith an equilibration buffer, and an antibody bound to the column wasthen eluted with an eluent (0.1% (v/v) HCl, 0.15 M NaCl). The peak areaof absorbance (O.D.280 nm) in the eluate was measured, and theconcentration was calculated according to the following equation:Antibody sample concentration (mg/mL)=(Peak area of antibodysample)/(Peak area of standard (human IgG1))×Concentration of standard(mg/mL)×Dilution ratio of sample.

(Example 3) Detection of Activation of ROBO4 Downstream Signal 3)-1Preparation of Reporter Vector Comprising Interleukin-8 (IL-8) PromoterRegion as Response Element

PCR reaction was performed with IL-8 promoter region DNA as a templateusing a primer set:

primer 17F: (SEQ ID NO: 101) 5′-ggtaccgataaggaacaaataggaag-3′ andprimer 17R: (SEQ ID NO: 102) 5′-gagctcagcttgtgtgctctgctgtc-3′.The obtained PCR product was incorporated to a pCR4Blunt-TOPO vector toprepare a cloning vector comprising IL-8 promoter region (−253 to −59)DNA. From the cloning vector, IL-8 promoter region (−253 to −59) DNA wascleaved off with KpnI and SacI and incorporated between KpnI and SacI ofa pGL4.15 vector (manufactured by Promega Corp.) to prepare a reportervector comprising the IL-8 promoter region as a response element. Thenucleotide sequence of IL-8 promoter region (−253 to −59) DNA is shownin SEQ ID NO: 29.

3)-2 Reporter Vector Comprising Nuclear Factor-κB (NF-κB), InterferonGamma Activation Sequence (GAS), Interferon Stimulated Response Element(ISRE), Transfection Grade T Cell Factor (TCF) as Response Element

pGL4.32[luc2P/NF-κB-RE/Hygro] Vector (manufactured by Promega Corp.),pGAS-TA-Luc Vector (manufactured by Takara Bio Inc.), pISRE-TA-LucVector (manufactured by Takara Bio Inc.), and TOPflash (manufactured byMillipore Corp.) were respectively used as reporter vectors comprisingNF-κB, GAS, ISRE, or TCF as a response element. Alternatively, responsesequence-free pTA-Luc Vector (manufactured by Takara Bio Inc.) was usedas a negative control. pRL-TK Vector (manufactured by Takara Bio Inc.)was used as an internal control.

3)-3 Analysis of Signal Varying in Cell Transiently Expressing HumanROBO4

HEK293 cells were inoculated at a concentration of 2×10⁴ cells/well to a96-well plate (coated with collagen I; manufactured by Asahi Glass Co.,Ltd.) and cultured overnight in a DMEM medium containing 10% FBS at 37°C. under 5% CO₂ conditions. On the next day, the HEK293 cells weretransfected with pCI-ROBO4, pCI-ROBO4-ΔC, or a negative control pCI-mockand each of the reporter vectors shown in 3)-1 and 3)-2 using FuGene6Transfection Reagent, and further cultured overnight at 37° C. under 5%CO₂ conditions. On the next day, the firefly luciferase and Renillaluciferase activities of each well were determined as luminescenceintensity in a plate reader (Mithras; manufactured by BertholdTechnologies GmbH & Co, KG) using Dual-Glo Luciferase Assay System(manufactured by Promega Corp.), and the reporter activity of each wellwas calculated according to the following equation: Reporteractivity=Firefly luciferase activity-derived luminescenceintensity/Renilla luciferase activity-derived luminescence intensity. Asa result, only the IL-8 promoter activity was increased in thepCI-hROBO4-transfected cells compared with the negative controlpCI-mock-transfected cells (FIG. 1). Moreover, this increase in the IL-8promoter activity detected in the pCI-hROBO4-transfected cells wasdrastically attenuated in the cells transfected with pCI-hROBO4-ΔC(intracellular region deletion mutant of hROBO4), demonstrating that theincrease in the IL-8 promoter activity detected in thepCI-hROBO4-transfected cells requires the intracellular region of ROBO4(FIG. 2). Accordingly, the increase in the IL-8 promoter activity in thepCI-hROBO4-transfected cells demonstrated that the activation of theROBO4 downstream signal was detected.

(Example 4) Properties of MAb1 4)-1 Activation of ROBO4 DownstreamSignal by MAb1

The pCI-hROBO4-transfected cells or the pCI-mock-transfected cellsprepared in 3)-3 were cultured overnight. On the next day, eachanti-ROBO4 antibody (MAb1, MAb2, MAb3, or MAb4) or a negative controlMouse IgG1 (manufactured by R&D systems, Inc.) was added thereto atconcentrations of 0, 0.3125, 1.25, 5, and 20 μg/mL or 0, 0.25, 1, 4, and16 μg/mL, and the cells were cultured at 37° C. for 5 hours under 5% CO₂conditions. Then, the firefly luciferase and Renilla luciferaseactivities of each well were determined as luminescence intensity in aplate reader (Mithras) using Dual-Glo Luciferase Assay System(manufactured by Promega Corp.), and the reporter activity of each wellwas calculated according to the following equation: Reporteractivity=Firefly luciferase activity-derived luminescenceintensity/Renilla luciferase activity-derived luminescence intensity. Asa result, the negative control mouse IgG did not influence the IL-8promoter activity in the cells transiently expressing human ROBO4,whereas MAb1 increased the IL-8 promoter activity (FIG. 3). As in MAb1,MAb2 also increased the IL-8 promoter activity, whereas MAb3 or MAb4 didnot increase the IL-8 promoter activity (FIG. 4). In the pCI-mock cells,MAb1 or MAb2 did not increase the IL-8 promoter activity. These resultsdemonstrated that MAb1 activated the downstream signal of ROBO4 and notall antibodies against ROBO4 activated the downstream signal of ROBO4.

MAb3 and MAb4 that were confirmed not to increase the activity of theROBO4 downstream signal were evaluated for promoter activity in thepresence of cross-linking antibodies (AffiPure Goat Anti-Mouse IgG FcFragment Specific, Cat NO. 115-005-071, Jackson ImmunoResearch) (twocross-linking antibody molecules with respect to one molecule of MAb3 orMAb4). As a result, increase in the promoter activity was observed forboth the antibodies.

4)-2 HUVEC Migration Test

HUVEC (manufactured by KURABO INDUSTRIES LTD.) was cultured overnight inHuMedia-EB2 (manufactured by KURABO INDUSTRIES LTD.) containing 0.1% BSAat 37° C. under 5% CO₂ conditions and then adjusted to 4×10⁵ cells/mLwith HuMedia-EB2 containing 0.1% BSA. 0.25 mL of the cell suspensionhaving a concentration of 4×10⁵ cells/mL was added to the upper layer ofa chamber in BD Falcon FluoroBlok 24 multi-well insert system (Poresize: 8 μm) having a gelatin-coated membrane. Then, HuMedia-EB2containing 0.1% BSA and 10 ng/mL human VEGF165 (manufactured byPeproTech Inc.) or human bFGF (BD Biosciences) and 2 μg/mL Mouse IgG2aor each anti-ROBO4 antibody (MAb1, MAb2, MAb3, or MAb4) was added to thelower layer of the chamber. After incubation at 37° C. for 2 to 3 hoursunder 5% CO₂ conditions, HUVEC that migrated to the lower layer wasstained with HuMedia-EB2 containing 4 μg/mL Calcein-AM (manufactured byLife Technologies Corp.) for 15 minutes. Then, the fluorescenceintensity (excitation wavelength/fluorescence wavelength: 485 nm/538 nm)of each well was measured using a plate reader (FlexStation; MolecularDevices, LLC.), and the amount of migrating cells in each well wascalculated according to the following equation: Amount of migratingcells=Fluorescence intensity of HUVEC-supplemented well−Fluorescenceintensity of HUVEC-unsupplemented well. As a result, MAb1 suppressed themigration of HUVEC induced by VEGF or bFGF (FIG. 5). As in MAb1, MAb2also suppressed the migration of HUVEC induced by bFGF, whereas MAb3 orMAb4 did not suppress the cell migration (FIG. 6). These resultsdemonstrated that the increase in the IL-8 promoter activity by theanti-ROBO4 antibody correlated with the suppressive activity againstHUVEC migration.

4)-3 Cross-Species Reactivity

4)-3-1 Preparation of Antigen Gene-Expressing Cell

HEK293 cells were inoculated at a concentration of 1.5×10⁶ cells/dish toa 60-mm dish (manufactured by Corning Inc.) and cultured overnight in aDMEM medium containing 10% FBS at 37° C. under 5% CO₂ conditions. On thenext day, the HEK293 cells were transfected with pCI-hROBO4, pCI-mROBO4,pCI-raROBO4, pCI-FLAG-cynoROBO4-1, pCI-FLAG-cynoROBO4-2, pCI-hROBO1,pCI-hROBO2, or pCI-hROBO3 using FuGENE6 Transfection Reagent and furthercultured overnight at 37° C. under 5% CO₂ conditions. On the next day,the expression vector-transfected cells were treated with TrypLE Express(manufactured by Life Technologies Corp.), washed with PBS containing 5%FBS, and then suspended in PBS containing 5% FBS. The obtained cellsuspension was used in flow cytometry analysis.

4)-3-2 Flow Cytometry Analysis

Each cell suspension prepared in 4)-3-1 was centrifuged, and thesupernatant was removed. Then, MAb1 or a negative control Mouse IgG2awas added at a concentration of 10 μg/mL to 2×10⁵ expressionvector-transfected cells, and the resulting suspension was left standingat 4° C. for 1 hour. The cells were washed once with PBS containing 5%FBS. Then, Anti-Mouse IgG FITC conjugate diluted 1000-fold with PBScontaining 5% FBS was added thereto, and the resulting suspension wasleft standing at 4° C. for 1 hour. The cells were washed with threetimes with PBS containing 5% FBS, then resuspended in PBS containing 5%FBS, and subjected to detection using a flow cytometer (BD FACSCalibur).Data was analyzed using Flowjo. The histogram of FITC fluorescenceintensity was prepared. The antibody was determined to bind in across-species manner when the histogram of MAb1 was shifted to astronger fluorescence intensity region compared with the histogram ofthe negative control Mouse IgG2a. The results of cross-speciesreactivity study demonstrated that MAb1 did not bind to mouse ROBO4 orrat ROBO4, but bound to human ROBO4 and cynomolgus monkey ROBO4 (FIG.7).

4)-4 Binding Specificity

4)-4-1 Preparation of Antigen Gene-Expressing Cell

HEK293 cells were inoculated at a concentration of 1.2×10⁶ cells/dish or1.5×10⁶ cells/dish to a 60-mm dish (manufactured by Corning Inc.) andcultured overnight in a DMEM medium containing 10% FBS at 37° C. under5% CO₂ conditions. On the next day, the HEK293 cells were transfectedwith pCI-hROBO4, pCI-FLAG-hROBO1, pCI-hROBO2, or pCI-hROBO3 usingFuGENE6 Transfection Reagent and further cultured overnight at 37° C.under 5% CO₂ conditions. On the next day, the expressionvector-transfected cells were treated with TrypLE Express (manufacturedby Life Technologies Corp.), washed with PBS containing 5% FBS, and thensuspended in PBS containing 5% FBS. The obtained cell suspension wasused in flow cytometry analysis.

4)-4-2 Flow Cytometry Analysis

Each cell suspension prepared in 4)-4-1 was centrifuged, and thesupernatant was removed. Then, MAb1, a positive control Human ROBO4Antibody (manufactured by R&D systems, Inc.), Monoclonal ANTI-FLAG M2antibody produced in mouse (manufactured by Sigma-Aldrich Corp.), HumanROBO2 Antibody (manufactured by R&D systems, Inc.), or MonoclonalAnti-human ROBO3 Antibody (manufactured by R&D systems, Inc.), or anegative control Mouse IgG1 or Mouse IgG2a was added at a concentrationof 10 μg/mL to 2×10⁵ expression vector-transfected cells, and theresulting suspension was left standing at 4° C. for 1 hour. The cellswere washed once with PBS containing 5% FBS. Then, Anti-Mouse IgG FITCconjugate diluted 1000-fold with PBS containing 5% FBS was addedthereto, and the resulting suspension was left standing at 4° C. for 1hour. The cells were washed with three times with PBS containing 5% FBS,then resuspended in PBS containing 5% FBS, and subjected to detectionusing a flow cytometer (BD FACSCalibur). Data was analyzed using Flowjo.The histogram of FITC fluorescence intensity was prepared. The antibodywas determined to bind in a specific manner when the histogram of MAb1was shifted to a stronger fluorescence intensity region compared withthe histogram of the negative control Mouse IgG1 or Mouse IgG2a. Theresults demonstrated that MAb1 did not bind to hROBO1, hROBO2, or hROBO3and specifically bound to hROBO4 (FIG. 8). In this context, hROBO4,hROBO1, hROBO2, and hROBO3 were each confirmed to be expressed on thecell membrane using the positive control antibody (FIG. 8).

4)-5 Epitope Determination

4)-5-1 Preparation of Antigen Gene-Expressing Cell

HEK293 cells were inoculated at a concentration of 1.5×10⁶ cells/dish toa 60-mm dish (manufactured by Corning Inc.) and cultured overnight in aDMEM medium containing 10% FBS at 37° C. under 5% CO₂ conditions. On thenext day, the HEK293 cells were transfected with pCI-FLAG-hROBO4-28,pCI-FLAG-hROBO4-46, pCI-FLAG-hROBO4-132, pCI-FLAG-hROBO4-210,pCI-FLAG-hROBO4-225, pCI-FLAG-hROBO4-341 using FuGENE6 TransfectionReagent and further cultured overnight at 37° C. under 5% CO₂conditions. On the next day, the expression vector-transfected cellswere treated with TrypLE Express (manufactured by Life TechnologiesCorp.), washed with PBS containing 5% FBS, and then suspended in PBScontaining 5% FBS. The obtained cell suspension was used in flowcytometry analysis.

4)-5-2 Flow Cytometry Analysis

Each cell suspension prepared in 4)-5-1 was centrifuged, and thesupernatant was removed. Then, MAb1, a positive control MonoclonalANTI-FLAG M2 antibody produced in mouse, or a negative control MouseIgG2a was added at a concentration of 10 μg/mL to 2×10⁵ expressionvector-transfected cells, and the resulting suspension was left standingat 4° C. for 1 hour. The cells were washed once with PBS containing 5%FBS. Then, Anti-Mouse IgG FITC conjugate diluted 1000-fold with PBScontaining 5% FBS was added thereto, and the resulting suspension wasleft standing at 4° C. for 1 hour. The cells were washed with threetimes with PBS containing 5% FBS, then resuspended in PBS containing 5%FBS, and subjected to detection using a flow cytometer (BD FACSCalibur;BD Biosciences). Data was analyzed using Flowjo. The histogram of FITCfluorescence intensity was prepared. The antibody was determined to bindto a cell when the histogram of MAb1 was shifted to a strongerfluorescence intensity region compared with the histogram of thenegative control Mouse IgG2a. The results demonstrated that MAb1 boundto the pCI-FLAG-hROBO4-28-, pCI-FLAG-hROBO4-46-, orpCI-FLAG-hROBO4-132-transfected cell and did not bind to thepCI-FLAG-hROBO4-210-, pCI-FLAG-hROBO4-225-, orpCI-FLAG-hROBO4-341-transfected cell. Thus, MAb1 was shown to recognizethe amino acid sequence of Nos. 132 to 209 in human ROBO4 shown in SEQID NO: 2 (FIG. 9). In this context, each intracellular region/domaindeletion variant was confirmed to be expressed on the cell membraneusing the positive control anti-FLAG antibody (FIG. 9).

4)-6 Drug Efficacy Evaluation in Monkey Models with Laser-InducedChoroidal Neovascularization

4)-6-1 Anesthesia

Medetomidine hydrochloride (manufactured by Nippon Zenyaku Kogyo Co.,Ltd.) was intramuscularly injected at a dose of 0.04 mg/kg to eachcynomolgus monkey. 15 minutes thereafter, ketamine hydrochloride(manufactured by Daiichi Sankyo Co., Ltd.) was intramuscularly injectedthereto at a dose of 15 mg/kg.

4)-6-2 Model Preparation

Each cynomolgus monkey anesthetized in 4)-6-1 was retained in a monkeychair. 4% Xylocaine eye drops (manufactured by AstraZeneca plc) wereapplied to both eyes for anesthetic/analgesic treatment of eye surface.5 mg/mL tropicamide-5 mg/mL phenylephrine hydrochloride mixture eyedrops (manufactured by Santen Pharmaceutical Co., Ltd.) were applied tothe eyes for mydriasis. The macular region of the retina was thermallydamaged by laser irradiation (quantity of heat irradiated: 350-500 mW,irradiation time: 0.1 seconds, spot size: 50 μm, the number of spots: 6or 9 spots) using a green laser photocoagulator OcuLight GLx(manufactured by Iridex Corp.).

4)-6-3 Administration of Test Substance

At day 7 after the model preparation, a 33G Nanopass needle was insertedinto the vitreous body from the conjunctiva, and 50 μL of vehicle or13.2 mg/mL MAb1 was injected thereto over 2 minutes using a 100-μLHamilton syringe via a PE20 polyethylene tube. The test was conducted oneach group involving 4 eyes. After completion of the administration,0.5% levofloxacin hydrate eye drops (manufactured by SantenPharmaceutical Co., Ltd.) were applied to the eyes.

4)-6-4 Drug Efficacy Evaluation

At days 7, 14, and 21 after the model preparation, the ocular fundus wasphotographed by a routine method using a hybrid fundus camera CX-1(manufactured by Canon Inc.) under anesthesia. Then, 10% fluorescein wasintravenously injected thereto at a dose of 0.1 mL/Kg. After completionof the intravenous injection of fluorescein, fluorescent angiography wasperformed every 1 minute up to 6 minutes later. The image data wasstored, and the area of a site at which fluorescein accumulated wascalculated using an image analyzer (WinRoof, manufactured by MitaniCorp.). The amount of blood vessels newly formed was calculatedaccording to the following equation: Amount of blood vessels newlyformed=Area of site at which fluorescein accumulated at day 21 aftermodel preparation−Area of site at which fluorescein accumulated at day 7after model preparation. As a result of comparing the amount of bloodvessels newly formed between the vehicle-administered group and theMAb1-administered group, three out of the four eyes in theMAb1-administered group were confirmed to decrease the amount of bloodvessels newly formed, though no difference was confirmed in the amountof blood vessels newly formed between the remaining one eye and thevehicle group. This means that the administration of MAb1 suppressedlaser-induced choroidal neovascularization (FIG. 10).

(Example 5) Cloning and Sequencing of MAb1 cDNA 5)-1 Determination ofN-Terminal Amino Acid Sequences of MAb1 Heavy and Light Chains

In order to determine the N-terminal amino acid sequences of the heavyand light chains of MAb1, MAb1 purified in Example 2)-8 was separated bySDS-PAGE. The proteins thus separated in the gel were transferred fromthe gel to a Sequi-Blot PVDF membrane (Bio-Rad Laboratories, Inc.),which was in turn washed with a washing buffer (25 mM NaCl, 10 mM sodiumborate buffer, pH 8.0), then stained by dipping for 5 minutes in astaining solution (50% methanol, 20% acetic acid, 0.05% CoomassieBrilliant Blue), and then decolorized with 90% methanol. Band portionscorresponding to the heavy chain (band with smaller mobility) and thelight chain (band with larger mobility) visualized on the PVDF membranewere cleaved off. Their respective N-terminal amino acid sequences wereidentified according to the automatic Edman method (see Edman et al.,(1967) Eur. J. Biochem. 1, 80) using Procise cLC protein sequencer Model492cLC (Applied Biosystems, Inc.). As a result, the N-terminal aminoacid sequence of the band corresponding to the heavy chain of MAb1 wasEVQLVESGGGLVKPGGSLKL (SEQ ID NO: 103), and the N-terminal amino acidsequence of the band corresponding to the light chain wasDAVMTQTPLSLPVSL (SEQ ID NO: 104).

5)-2 Preparation of mRNA from MAb1-Producing Hybridoma

In order to clone heavy chain- and light chain-encoding cDNAs of MAb1,mRNA was prepared from the MAb1-producing hybridoma using mRNA Isolationkit (Roche Applied Science).

5)-3 Cloning and Sequencing of MAb1 cDNA

Several oligonucleotide primers hybridizing to the 5′-terminal sequenceof the coding region of the antibody gene and the stop codon-containing3′-terminal sequence thereof, respectively, were synthesized withreference to the N-terminal amino acid sequences of the heavy and lightchains determined based on the γ1 and κ isotypes of the MAb1 heavy andlight chains, respectively (Examples 2)-7) and 5-1), and the antibodyamino acid sequence database prepared by Kabat et al. (see Strausberg,R. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99. 16899-16903, andKabat, E. A., et al. (1991) in Sequences of Proteins of ImmunologicalInterest Vol. I and II, U. S. Department of Health and Human Services).Heavy chain- and light chain-encoding cDNAs were amplified using themRNA prepared in 5)-2 and TaKaRa One Step RNA PCR Kit (AMV) (Takara BioInc.). As a result, the heavy chain- and light chain-encoding cDNAs ofthe antibody were successfully amplified using the following primer set:

primer set for the heavy chain LYHF6: (SEQ ID NO: 105)5′-cctcaccatgaactttgg-3′ G1EVR1: (SEQ ID NO: 106)5′-aagatatcttatttaccaggagagtgggagag-3′ primer set for the light chainMK19EIF1: (SEQ ID NO: 107) 5′-aagaattcatgaagttgcctgttagg-3′ KEVR1:(SEQ ID NO: 108) 5′-aagatatcttaacactcattcctgttgaagct-3′.

The heavy and light chain cDNAs amplified by PCR were separately clonedusing pEF6/V5-His TOPO TA Expression Kit (Invitrogen Corp.). Thenucleotide sequences of the cloned cDNAs encoding the respectivevariable regions of the heavy and light chains were determined using agene sequencer (“ABI PRISM 3700 DNA Analyzer; Applied Biosystems” or“Applied Biosystems 3730xl Analyzer; Applied Biosystems”). Thesequencing reaction was performed using GeneAmp 9700 (AppliedBiosystems, Inc.).

The determined nucleotide sequence of cDNA encoding the heavy chainvariable region of MAb1 is shown in SEQ ID NO: 30, and its amino acidsequence is shown in SEQ ID NO: 31. The nucleotide sequence of cDNAencoding the light chain variable region of the mouse antibody MAb1 isshown in SEQ ID NO: 32, and its amino acid sequence is shown in SEQ IDNO: 33.

(Example 6) Preparation of Chimeric MAb1 (cMAb1) 6)-1 Preparation ofExpression Vectors pCMA-LK, pCMA-G1, and pCMA-G2

6)-1-1 Construction of Chimeric and Humanized Light Chain ExpressionVector pCMA-LK

A plasmid pcDNA3.3-TOPO/LacZ (Invitrogen Corp.) was digested withrestriction enzymes XbaI and PmeI, and the obtained fragment ofapproximately 5.4 kb was ligated to a DNA fragment comprising a DNAsequence (SEQ ID NO: 34) encoding a human κ chain secretory signal and ahuman κ chain constant region using In-Fusion Advantage PCR cloning kit(Clontech Laboratories, Inc.) to prepare pcDNA3.3/LK.

PCR was performed with pcDNA3.3/LK as a template using a primer setshown below. The obtained fragment of approximately 3.8 kb wasphosphorylated and then self-ligated to construct, from pcDNA3.3/LK, achimeric and humanized light chain expression vector pCMA-LK having asignal sequence-encoding sequence, a cloning site, and a human κ chainconstant region-encoding sequence downstream of a CMV promoter.

Primer set 3.3-F1: (SEQ ID NO: 109) 5′-tataccgtcgacctctagctagagcttggc-3′3.3-R1: (SEQ ID NO: 110) 5′-gctatggcagggcctgccgccccgacgttg-3′.6)-1-2 Construction of Chimeric and Humanized IgG1-Type Heavy ChainExpression Vector pCMA-G1

pCMA-LK was digested with XbaI and PmeI to remove a sequence encoding aκ chain secretory signal and a human κ chain constant region. Theresulting DNA fragment was ligated to a DNA fragment comprising a DNAsequence (SEQ ID NO: 35) encoding the amino acids of a human heavy chainsignal sequence and a human IgG1 constant region using In-FusionAdvantage PCR cloning kit (Clontech Laboratories, Inc.) to construct achimeric and humanized IgG1-type heavy chain expression vector pCMA-G1having a signal sequence-encoding sequence, a cloning site, and a humanIgG1 heavy chain constant region-encoding sequence downstream of a CMVpromoter.

6)-1-3 Construction of Chimeric and Humanized IgG2-Type Heavy ChainExpression Vector pCMA-G2

pCMA-LK was digested with XbaI and PmeI to remove a sequence encoding aκ chain secretory signal and a human κ chain constant region. Theresulting DNA fragment was ligated to a DNA fragment comprising a DNAsequence (SEQ ID NO: 36) encoding the amino acids of a human heavy chainsignal sequence and a human IgG2 constant region using In-FusionAdvantage PCR cloning kit (Clontech Laboratories, Inc.) to construct achimeric and humanized IgG2-type heavy chain expression vector pCMA-G2having a signal sequence-encoding sequence, a cloning site, and a humanIgG2 heavy chain constant region-encoding sequence downstream of a CMVpromoter.

6)-2 Construction of Chimeric MAb1 Light Chain Expression Vector

A site comprising cDNA encoding the light chain variable region wasamplified with cDNA encoding the light chain variable region of MAb1 asa template using KOD-Plus-(TOYOBO CO., LTD.) and a primer set shownbelow, and inserted into a restriction enzyme BsiWI-cleaved site of thegeneral-purpose vector pCMA-LK for chimeric and humanized antibody lightchain expression using In-Fusion Advantage PCR cloning kit (ClontechLaboratories, Inc.) to construct a chimeric MAb1 light chain expressionvector. The obtained expression vector was designated as “pCMA-LK/MAb1L”. The nucleotide sequence encoding the chimeric MAb1 light chain isshown in SEQ ID NO: 37, and its amino acid sequence is shown in SEQ IDNO: 38. Nucleotide Nos. 1 to 60 of SEQ ID NO: 37 represent thenucleotide sequence encoding the signal sequence. Nucleotide Nos. 61 to402 thereof represent the nucleotide sequence encoding the variableregion. Nucleotide Nos. 403 to 717 thereof represent the nucleotidesequence encoding the constant region. Amino acid Nos. 1 to 20 of SEQ IDNO: 38 represent the amino acids of the signal sequence. Amino acid Nos.21 to 134 thereof represent the amino acids of the variable region.Amino acid Nos. 135 to 239 represent the amino acids of the constantregion.

Primer set for the light chain MAb1 LF: (SEQ ID NO: 111)5′-tctccggcgcgtacggcgatgctgtgatgacccaaactccact ctcc-3′ MAb1 LR:(SEQ ID NO: 112) 5′-ggagggggcggccacagcccgtttgatttccagcttggtgcc tcc-3′

6)-3 Construction of Chimeric MAb1 IgG1-Type Heavy Chain ExpressionVector

A site comprising cDNA encoding the heavy chain variable region wasamplified with cDNA encoding the heavy chain variable region of MAb1 asa template using KOD-Plus-(TOYOBO CO., LTD.) and a primer set shownbelow, and inserted into a restriction enzyme BsiWI-cleaved site of thechimeric and humanized IgG1-type heavy chain expression vector pCMA-G1using In-Fusion Advantage PCR cloning kit (Clontech Laboratories, Inc.)to construct a chimeric MAb1 IgG1-type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G1/MAb1 H”. Thenucleotide sequence encoding the chimeric MAb1 IgG1-type heavy chain isshown in SEQ ID NO: 39, and its amino acid sequence is shown in SEQ IDNO: 40. Nucleotide Nos. 1 to 57 of SEQ ID NO: 39 represent thenucleotide sequence encoding the signal sequence. Nucleotide Nos. 58 to411 thereof represent the nucleotide sequence encoding the variableregion. Nucleotide Nos. 412 to 1401 thereof represent the nucleotidesequence encoding the constant region. Amino acid Nos. 1 to 19 of SEQ IDNO: 40 represent the amino acid sequence of the signal sequence. Aminoacid Nos. 20 to 137 thereof represent the amino acid sequence of thevariable region. Amino acid Nos. 138 to 467 represent the amino acidsequence of the constant region.

Primer set for the IgG1-type heavy chain MAb1 HF: (SEQ ID NO: 113)5′-cagatgggtgctgagcgaagtgcagctggtggagt ctgggggag-3′ MAb1 H1R:(SEQ ID NO: 114) 5′-ttggtggaggctgagctgactgtgagagtggtgc cgtggccccag-3′.

6)-4 Construction of Chimeric MAb1 IgG2-Type Heavy Chain ExpressionVector

A site comprising cDNA encoding the heavy chain variable region wasamplified with cDNA encoding the heavy chain variable region of MAb1 asa template using KOD-Plus-(TOYOBO CO., LTD.) and a primer set shownbelow, and inserted into a restriction enzyme BsiWI-cleaved site of thechimeric and humanized IgG2-type heavy chain expression vector pCMA-G2using In-Fusion Advantage PCR cloning kit (Clontech Laboratories, Inc.)to construct a chimeric MAb1 IgG2-type heavy chain expression vector.The obtained expression vector was designated as “pCMA-G2/MAb1”. Thenucleotide sequence encoding the chimeric MAb1 IgG2-type heavy chain isshown in SEQ ID NO: 41, and its amino acid sequence is shown in SEQ IDNO: 42.

Primer set for the IgG2-type heavy chain MAb1 HF: (SEQ ID NO: 113)5′-cagatgggtgctgagcgaagtgcagaggtggagtct gggggag-3′ MAb1 2R:(SEQ ID NO: 115) 5′-ttggtgctggctgagctgactgtgagagtggtgccg tggccccag-3′

(Example 7) Preparation of IgG1-Type Chimeric MAb1 Antibody andIgG2-Type Chimeric MAb1 Antibody 7)-1 Production of IgG1-Type ChimericMAb1 Antibody and IgG2-Type Chimeric MAb1 Antibody

FreeStyle 293-F cells (Invitrogen Corp.) were subcultured and culturedaccording to the manual. 1.2×10⁹ cells of FreeStyle 293-F cells(Invitrogen Corp.) at the logarithmic growth phase were inoculated to a3 L Fernbach Erlenmeyer Flask (Corning Inc.), diluted with FreeStyle 293expression medium (Invitrogen Corp.) into 1.0×10⁶ cells/mL, and thenshake-cultured in an 8% CO₂ incubator at 90 rpm at 37° C. for 1 hour.3.6 mg of polyethyleneimine (Polysciences, Inc., #24765) was dissolvedin 20 mL of Opti-Pro SFM medium (Invitrogen Corp.). Next, an H chainexpression vector (0.4 mg) and an L chain expression vector (0.8 mg)prepared using PureLink HiPure Plasmid kit (Invitrogen Corp.) weresuspended in 20 mL of Opti-Pro SFM medium (Invitrogen Corp.). 20 mL ofthe expression vector/Opti-Pro SFM mixture solution was added to 20 mLof the polyethyleneimine/Opti-Pro SFM mixture solution, and the mixturewas gradually stirred, further left for 5 minutes, and then added to theFreeStyle 293-F cells. The cells were shake-cultured in an 8% CO₂incubator at 90 rpm at 37° C. for 7 days, and the obtained culturesupernatant was filtered through Disposable Capsule Filter (Advantec,#CCS-045-E1H).

IgG1-type and IgG2-type chimeric MAb1 antibodies obtained by thecombinations between pCMA-G1/MAb1 H and pCMA-LK/MAb1 L and betweenpCMA-G2/MAb1 H and pCMA-LK/MAb1 L are abbreviated to “cMAb1-1” and“cMAb1-2”, respectively. The term “cMAb1” means both IgG1-type andIgG2-type chimeric MAb1 antibodies.

7)-2 Purification of cMAb1-1 and cMAb1-2

The culture supernatant obtained above in 7-1) was purified by rProteinA affinity chromatography (at 4-6° C.). A buffer replacement step afterthe rProtein A affinity chromatography purification was carried out atroom temperature. First, 1100 to 1200 ml of the culture supernatant wasapplied to Mab Select SuRe (manufactured by GE Healthcare Bio-SciencesCorp., HiTrap column; volume 1 mL×2 connected) equilibrated with PBS.The whole culture solution was placed in the column, and the column wasthen washed with PBS. Next, elution was performed with a 2 M argininehydrochloride solution (pH 4.0) to collect an antibody-containingfraction. The buffer in this fraction was replaced by PBS using adesalting column (manufactured by GE Healthcare Bio-Sciences Corp.,HiTrap Desalting column; volume 5 mL×2 connected). Finally, the fractionwas concentrated with Centrifugal UF Filter Device VIVASPIN20 (molecularweight cutoff: 30 K, Sartorius, at 4° C.) into an IgG concentration of20.0 mg/mL or higher and used as a purification sample.

7)-3 Properties of cMAb1-1 and cMAb1-2

7)-3-1 Activation of ROBO4 Downstream Signal

This test was conducted according to the method of 4)-1 except that:Human IgG (manufactured by Sigma-Aldrich Corp.) was used as a negativecontrol; and Human IgG, cMAb1-1, or cMAb1-2 was added at a finalconcentration of 0.313 μg/mL to a medium. As a result, the negativecontrol Human IgG did not influence the IL-8 promoter activity in thecells transiently expressing human ROBO4, whereas cMAb1-1 and cMAb1-2increased the IL-8 promoter activity (FIG. 11). These resultsdemonstrated that cMAb1-1 or cMAb1-2 activated the downstream signal ofROBO4, as in MAb1.

7)-3-2 HUVEC Migration Test

This test was conducted according to the method of 4)-2 except that:Human IgG was used as a negative control; and Human IgG, cMAb1-1, orcMAb1-2 was added at a final concentration of 0.5 μg/mL to a medium. Asa result, cMAb1-1 or cMAb1-2 suppressed the migration of HUVEC inducedby bFGF (FIG. 12). These results demonstrated that cMAb1-1 or cMAb1-2suppressed the migration of HUVEC, as in MAb1.

(Example 8) Design of Humanized Antibody of Mouse Anti-Human ROBO4Monoclonal Antibody MAb1 8)-1 Design of Humanized Version of MAb1

8)-1-1 Molecular Modeling of MAb1 Variable Region

The molecular modeling of the MAb1 variable regions was carried out by agenerally known homology modeling method (Methods in Enzymology, 203,121-153, (1991)). The primary sequences (available as three-dimensionalstructures induced from x-ray crystal structures) of humanimmunoglobulin variable regions registered in Protein Data Bank (Nuc.Acid Res. 35, D301-D303 (2007)) were compared with those of the MAb1variable regions thus determined. As a result, 3FFD was selected becauseof its highest sequence homology to the MAb1 heavy chain variable regionamong antibodies deficient in framework. Also, 1T66 was selected becauseof its highest sequence homology to the MAb1 light chain variableregion. The three-dimensional structures of the framework regions wereprepared by obtaining a “framework model” from the combination of thecoordinates of 3FFD and 1T66 corresponding to the MAb1 heavy and lightchains. As for the CDRs of MAb1, CDRL1, CDRL2, CDRL3, CDRH1, and CDRH2were assigned to clusters 16A, 7A, 9A, 10A, and 10B, respectively,according to the classification of Thornton et al. (J. Mol. Biol., 263,800-815, (1996)). CDRH3 was classified into k(7)B using the H3 rule(FEBS letter 399, 1-8 (1996)). Subsequently, the typical conformation ofeach CDR was incorporated into the framework model.

Finally, energy calculation for excluding disadvantageous interatomiccontact was conducted to obtain a molecular model likely to be the MAb1variable regions in terms of energy. These procedures were performedusing a commercially available protein three-dimensional structureprediction program Prime and coordinate searching program MacroModel(Schrodinger, LLC).

8)-1-2 Design of Humanized MAb1 Amino Acid Sequence

Each humanized MAb1 antibody (hMAb1) was constructed by a generallyknown CDR grafting method (Proc. Natl. Acad. Sci. USA 86, 10029-10033(1989)). Two different acceptor antibodies were selected on the basis ofthe homology of amino acids in framework regions. The sequences of theMAb1 framework regions were compared with all human frameworks in theKabat antibody amino acid sequence database (Nuc. Acid Res. 29, 205-206(2001)). As a result, a B3 antibody was selected as an acceptor becauseof its 83% sequence homology to the framework regions. The amino acidresidues of the B3 framework regions were aligned with those of MAb1 toidentify the positions of different amino acids used. These positions ofthe residues were analyzed using the three-dimensional model of MAb1constructed above. Then, donor residues to be grafted onto the acceptorwere selected according to the criteria given by Queen et al. (Proc.Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Some selected donorresidues were integrated into the acceptor antibody to construct thesequence of humanized MAb1 as described in Examples below. Also, hMAb1variants were constructed by the substitution of 1 to 3 amino acidresidue(s) in each CDR of hMAb1 with other amino acid residues, asdescribed in Examples below.

8)-2 Humanization of cMAb1 Heavy Chain

8)-2-1 hMAb1-H1-Type Heavy Chain:

A humanized MAb1 heavy chain designed by replacing amino acid No. 32(lysine) with glutamine, amino acid No. 38 (lysine) with arginine, aminoacid No. 59 (threonine) with alanine, amino acid No. 61 (glutamic acid)with glycine, amino acid No. 63 (arginine) with glycine, amino acid No.95 (glutamic acid) with lysine, amino acid No. 103 (serine) withasparagine, amino acid No. 107 (serine) with alanine, amino acid No. 112(methionine) with valine, amino acid No. 114 (phenylalanine) withtyrosine, amino acid No. 129 (histidine) with glutamine, amino acid No.132 (threonine) with leucine, and amino acid No. 133 (leucine) withvaline, in the cMAb1-2 heavy chain represented by SEQ ID NO: 42 wasdesignated as a “hMAb1-H1-type heavy chain”.

The amino acid sequence of the hMAb1-H1-type heavy chain is shown in SEQID NO: 56. A sequence consisting of amino acid residues 1 to 19, asequence consisting of amino acid residues 20 to 137, and a sequenceconsisting of amino acid residues 138 to 463 in the amino acid sequenceof SEQ ID NO: 56 correspond to a signal sequence, a heavy chain variableregion, and a heavy chain constant region, respectively. A sequenceconsisting of amino acid residues 50 to 54, a sequence consisting ofamino acid residues 69 to 85, and a sequence consisting of amino acidresidues 118 to 126 in the amino acid sequence of SEQ ID NO: 56correspond to a CDRH1 sequence, a CDRH2 sequence, and a CDRH3 sequence,respectively. A nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 56 is shown in SEQ ID NO: 55. A sequence consisting ofnucleotides 1 to 57, a sequence consisting of nucleotides 58 to 411, anda sequence consisting of nucleotides 412 to 1389 in the nucleotidesequence of SEQ ID NO: 55 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 55 and the aminoacid sequence of SEQ ID NO: 56 are also shown in FIGS. 31 and 32,respectively.

8)-2-2 hMAb1-H2-Type Heavy Chain:

A humanized MAb1 heavy chain designed by replacing amino acid No. 32(lysine) with glutamine, amino acid No. 38 (lysine) with arginine, aminoacid No. 59 (threonine) with alanine, amino acid No. 61 (glutamic acid)with glycine, amino acid No. 63 (arginine) with glycine, amino acid No.72 (asparagine) with glutamine, amino acid No. 95 (glutamic acid) withlysine, amino acid No. 103 (serine) with asparagine, amino acid No. 107(serine) with alanine, amino acid No. 112 (methionine) with valine,amino acid No. 114 (phenylalanine) with tyrosine, amino acid No. 129(histidine) with glutamine, amino acid No. 132 (threonine) with leucine,and amino acid No. 133 (leucine) with valine, in the cMAb1-2 heavy chainrepresented by SEQ ID NO: 42 was designated as a “hMAb1-H2-type heavychain”.

The amino acid sequence of the hMAb1-H2-type heavy chain is shown in SEQID NO: 58. A sequence consisting of amino acid residues 1 to 19, asequence consisting of amino acid residues 20 to 137, and a sequenceconsisting of amino acid residues 138 to 463 in the amino acid sequenceof SEQ ID NO: 58 correspond to a signal sequence, a heavy chain variableregion, and a heavy chain constant region, respectively. A sequenceconsisting of amino acid residues 50 to 54, a sequence consisting ofamino acid residues 69 to 85, and a sequence consisting of amino acidresidues 118 to 126 in the amino acid sequence of SEQ ID NO: 58correspond to a CDRH1 sequence, a CDRH2 sequence, and a CDRH3 sequence,respectively. A nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 58 is shown in SEQ ID NO: 57. A sequence consisting ofnucleotides 1 to 57, a sequence consisting of nucleotides 58 to 411, anda sequence consisting of nucleotides 412 to 1389 in the nucleotidesequence of SEQ ID NO: 57 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 57 and the aminoacid sequence of SEQ ID NO: 58 are also shown in FIGS. 33 and 34,respectively.

8)-2-3 hMAb1-H3-Type Heavy Chain:

A humanized MAb1 heavy chain designed by replacing amino acid No. 32(lysine) with glutamine, amino acid No. 38 (lysine) with arginine, aminoacid No. 59 (threonine) with alanine, amino acid No. 61 (glutamic acid)with glycine, amino acid No. 95 (glutamic acid) with lysine, amino acidNo. 103 (serine) with asparagine, amino acid No. 107 (serine) withalanine, amino acid No. 112 (methionine) with valine, amino acid No. 114(phenylalanine) with tyrosine, amino acid No. 129 (histidine) withglutamine, amino acid No. 132 (threonine) with leucine, and amino acidNo. 133 (leucine) with valine, in the cMAb1-2 heavy chain represented bySEQ ID NO: 42 was designated as a “hMAb1-H3-type heavy chain”.

The amino acid sequence of the hMAb1-H3-type heavy chain is shown in SEQID NO: 60. A sequence consisting of amino acid residues 1 to 19, asequence consisting of amino acid residues 20 to 137, and a sequenceconsisting of amino acid residues 138 to 463 in the amino acid sequenceof SEQ ID NO: 60 correspond to a signal sequence, a heavy chain variableregion, and a heavy chain constant region, respectively. A sequenceconsisting of amino acid residues 50 to 54, a sequence consisting ofamino acid residues 69 to 85, and a sequence consisting of amino acidresidues 118 to 126 in the amino acid sequence of SEQ ID NO: 60correspond to a CDRH1 sequence, a CDRH2 sequence, and a CDRH3 sequence,respectively. A nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 60 is shown in SEQ ID NO: 59. A sequence consisting ofnucleotides 1 to 57, a sequence consisting of nucleotides 58 to 411, anda sequence consisting of nucleotides 412 to 1389 in the nucleotidesequence of SEQ ID NO: 59 encode the signal sequence, the heavy chainvariable region sequence, and the heavy chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 59 and the aminoacid sequence of SEQ ID NO: 60 are also shown in FIGS. 35 and 36,respectively.

8)-2-4 hMAb1-H4-Type Heavy Chain:

A humanized MAb1 heavy chain designed by replacing amino acid No. 32(lysine) with glutamine, amino acid No. 38 (lysine) with arginine, aminoacid No. 59 (threonine) with alanine, amino acid No. 61 (glutamic acid)with glycine, amino acid No. 72 (asparagine) with glutamine, amino acidNo. 95 (glutamic acid) with lysine, amino acid No. 103 (serine) withasparagine, amino acid No. 107 (serine) with alanine, amino acid No. 112(methionine) with valine, amino acid No. 114 (phenylalanine) withtyrosine, amino acid No. 129 (histidine) with glutamine, amino acid No.132 (threonine) with leucine, and amino acid No. 133 (leucine) withvaline, in the cMAb1-2 heavy chain represented by SEQ ID NO: 42 wasdesignated as a “hMAb1-H4-type heavy chain”.

The amino acid sequence of the hMAb1-H4-type heavy chain is shown in SEQID NO: 62. A sequence consisting of amino acid residues 1 to 19, asequence consisting of amino acid residues 20 to 137, and a sequenceconsisting of amino acid residues 138 to 463 in the amino acid sequenceof SEQ ID NO: 62 correspond to a signal sequence, a heavy chain variableregion, and a heavy chain constant region, respectively. A sequenceconsisting of amino

acid residues 50 to 54, a sequence consisting of amino acid residues 69to 85, and a sequence consisting of amino acid residues 118 to 126 inthe amino acid sequence of SEQ ID NO: 62 correspond to a CDRH1 sequence,a CDRH2 sequence, and a CDRH3 sequence, respectively. A nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 62 is shown inSEQ ID NO: 61. A sequence consisting of nucleotides 1 to 57, a sequenceconsisting of nucleotides 58 to 411, and a sequence consisting ofnucleotides 412 to 1389 in the nucleotide sequence of SEQ ID NO: 61encode the signal sequence, the heavy chain variable region sequence,and the heavy chain constant region sequence, respectively. Thenucleotide sequence of SEQ ID NO: 61 and the amino acid sequence of SEQID NO: 62 are also shown in FIGS. 37 and 38, respectively.

8)-3 Humanization of MAb1 Light Chain

8)-3-1 hMAb1-L1-Type Light Chain:

A humanized MAb1 light chain designed by replacing amino acid No. 22(alanine) with isoleucine, amino acid No. 27 (threonine) with serine,amino acid No. 34 (serine) with threonine, amino acid No. 37 (asparticacid) with glutamic acid, amino acid No. 38 (glutamine) with proline,amino acid No. 62 (phenylalanine) with leucine, amino acid No. 84(leucine) with proline, amino acid No. 108 (phenylalanine) with valine,amino acid No. 112 (phenylalanine) with tyrosine, amino acid No. 125(glycine) with proline, amino acid No. 129 (leucine) with valine, aminoacid No. 130 (glutamic acid) with aspartic acid, and amino acid No. 134(alanine) with threonine, in the cMAb1 light chain represented by SEQ IDNO: 38 was designated as a “hMAb1-L1-type light chain”.

The amino acid sequence of the hMAb1-L1-type light chain is shown in SEQID NO: 64. A sequence consisting of amino acid residues 1 to 20, asequence consisting of amino acid residues 21 to 134, and a sequenceconsisting of amino acid residues 135 to 239 in the amino acid sequenceof SEQ ID NO: 64 correspond to a signal sequence, a light chain variableregion, and a light chain constant region, respectively. A sequenceconsisting of amino acid residues 44 to 59, a sequence consisting ofamino acid residues 75 to 81, and a sequence consisting of amino acidresidues 114 to 122 in the amino acid sequence of SEQ ID NO: 64correspond to a CDRL1 sequence, a CDRL2 sequence, and a CDRL3 sequence,respectively. A nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 64 is shown in SEQ ID NO: 63. A sequence consisting ofnucleotides 1 to 60, a sequence consisting of nucleotides 61 to 402, anda sequence consisting of nucleotides 403 to 717 in the nucleotidesequence of SEQ ID NO: 63 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 63 and the aminoacid sequence of SEQ ID NO: 64 are also shown in FIGS. 39 and 40,respectively.

8)-3-2 hMAb1-L2-Type Light Chain:

A humanized MAb1 light chain designed by replacing amino acid No. 22(alanine) with isoleucine, amino acid No. 27 (threonine) with serine,amino acid No. 34 (serine) with threonine, amino acid No. 37 (asparticacid) with glutamic acid, amino acid No. 38 (glutamine) with proline,amino acid No. 52 (serine) with glutamic acid, amino acid No. 54(glycine) with lysine, amino acid No. 56 (threonine) with leucine, aminoacid No. 62 (phenylalanine) with leucine, amino acid No. 84 (leucine)with proline, amino acid No. 108 (phenylalanine) with valine, amino acidNo. 112 (phenylalanine) with tyrosine, amino acid No. 125 (glycine) withproline, amino acid No. 129 (leucine) with valine, amino acid No. 130(glutamic acid) with aspartic acid, and amino acid No. 134 (alanine)with threonine, in the cMAb1 light chain represented by SEQ ID NO: 38was designated as a “hMAb1-L2-type light chain”.

The amino acid sequence of the hMAb1-L2-type light chain is shown in SEQID NO: 66. A sequence consisting of amino acid residues 1 to 20, asequence consisting of amino acid residues 21 to 134, and a sequenceconsisting of amino acid residues 135 to 239 in the amino acid sequenceof SEQ ID NO: 66 correspond to a signal sequence, a light chain variableregion, and a light chain constant region, respectively. A sequenceconsisting of amino acid residues 44 to 59, a sequence consisting ofamino acid residues 75 to 81, and a sequence consisting of amino acidresidues 114 to 122 in the amino acid sequence of SEQ ID NO: 66correspond to a CDRL1 sequence, a CDRL2 sequence, and a CDRL3 sequence,respectively. A nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 66 is shown in SEQ ID NO: 65. A sequence consisting ofnucleotides 1 to 60, a sequence consisting of nucleotides 61 to 402, anda sequence consisting of nucleotides 403 to 717 in the nucleotidesequence of SEQ ID NO: 65 encode the signal sequence, the light chainvariable region sequence, and the light chain constant region sequence,respectively. The nucleotide sequence of SEQ ID NO: 65 and the aminoacid sequence of SEQ ID NO: 66 are also shown in FIGS. 41 and 42,respectively.

(Example 9) Preparation of Humanized MAb1 9)-1 Construction of HumanizedMAb1 Heavy Chain Expression Vector

9)-1-1 Construction of hMAb1-H1-Type Heavy Chain Expression Vector

DNA comprising the gene encoding the hMAb1-H1-type heavy chain variableregion represented by amino acid Nos. 20 to 137 of SEQ ID NO: 56 wassynthesized (GeneArt artificial gene synthesis service) and cleaved witha restriction enzyme BlpI. The obtained DNA fragment was inserted to arestriction enzyme BlpI-cleaved site of the chimeric and humanizedIgG2-type heavy chain expression vector (pCMA-G2) to construct ahMAb1-H1-type heavy chain expression vector. The obtained expressionvector was designated as “pCMA-G2/hMAb1-H1”. The nucleotide sequence ofthe hMAb1-H1-type heavy chain is shown in SEQ ID NO: 55.

9)-1-2 Construction of hMAb1-H2-Type Heavy Chain Expression Vector

A hMAb1-H2-type heavy chain expression vector was constructed with thepCMA-G2/hMAb1-H1 constructed in 9)-1-1 as a template using a primer setshown below and QuikChange XL Site-Directed Mutagenesis Kit (AgilentTechnologies, Inc.). The obtained expression vector was designated as“pCMA-G1/hMAb1-H2”. The nucleotide sequence of the hMAb1-H2-type heavychain is shown in SEQ ID NO: 57, and its amino acid sequence is shown inSEQ ID NO: 58.

Primer set: H-N53Q-F: (SEQ ID NO: 116)5′-gggtggcaaccatcagccaaggcggcacctacacctac-3′ H-N53Q-R: (SEQ ID NO: 117)5′-gtaggtgtaggtgccgccttggctgatggttgccaccc-3′9)-1-3 Construction of hMAb1-H3-Type Heavy Chain Expression Vector

DNA comprising the gene encoding the hMAb1-H3-type heavy chain variableregion represented by amino acid Nos. 20 to 137 of SEQ ID NO: 60 wassynthesized (GeneArt artificial gene synthesis service) and cleaved witha restriction enzyme BlpI. The obtained DNA fragment was inserted to arestriction enzyme BlpI-cleaved site of the chimeric and humanizedIgG2-type heavy chain expression vector (pCMA-G2) to construct ahMAb1-H3-type heavy chain expression vector. The obtained expressionvector was designated as “pCMA-G2/hMAb1-H3”. The nucleotide sequence ofthe hMAb1-H3-type heavy chain is shown in SEQ ID NO: 59.

9)-1-4 Construction of hMAb1-H4-Type Heavy Chain Expression Vector

A hMAb1-H4-type heavy chain expression vector was constructed with thepCMA-G2/hMAb1-H3 constructed in 9)-1-3 as a template using a primer setshown below and QuikChange XL Site-Directed Mutagenesis Kit (AgilentTechnologies, Inc.). The obtained expression vector was designated as“pCMA-G1/hMAb1-H4”. The nucleotide sequence of the hMAb1-H4-type heavychain is shown in SEQ ID NO: 61, and its amino acid sequence is shown inSEQ ID NO: 62.

Primer set: H-N53Q-F: (SEQ ID NO: 116)5′-gggtggcaaccatcagccaaggcggcacctacacctac-3′ H-N53Q-R: (SEQ ID NO: 117)5′-gtaggtgtaggtgccgccttggctgatggttgccaccc-3′

9)-2 Construction of Humanized MAb1 Light Chain Expression Vector

9)-2-1 Construction of hMAb1-L1-Type Light Chain Expression Vector

DNA comprising the gene encoding the hMAb1-L1-type light chain variableregion represented by amino acid Nos. 21 to 134 of SEQ ID NO: 64 wassynthesized (GeneArt artificial gene synthesis service) and cleaved witha restriction enzyme BsiWI. The obtained DNA fragment was inserted to arestriction enzyme BsiWI-cleaved site of the general-purpose vector(pCMA-LK) for chimeric and humanized antibody light chain expression toconstruct a hMAb1-L1-type light chain expression vector. The obtainedexpression vector was designated as “pCMA-LK/hMAb1-L1”. The nucleotidesequence of the hMAb1-L1-type light chain is shown in SEQ ID NO: 63.

9)-2-2 Construction of hMAb1-L2-Type Light Chain Expression Vector

DNA fragments were obtained by PCR with the pCMA-LK/hMAb1-L1 constructedin 9)-2-1 as a template using KOD-Plus-(TOYOBO CO., LTD.) and each ofprimer sets A and B, and linked by overlap extension PCR using primerset C to prepare DNA comprising a gene encoding the hMAb1-L2-type lightchain. This DNA was cleaved with restriction enzymes XbaI and PmeI toobtain a DNA fragment, which was then inserted to a restriction enzymeXbaI/PmeI-cleaved site of the general-purpose vector (pCMA-LK) forchimeric and humanized antibody light chain expression to construct ahMAb1-L2-type light chain expression vector. The obtained expressionvector was designated as “pCMA-LK/hMAb1-L2”. The nucleotide sequence ofthe hMAb1-L2-type light chain is shown in SEQ ID NO: 65, and its aminoacid sequence is shown in SEQ ID NO: 66.

Primer set A L inf-F: (SEQ ID NO: 118)5′-gcctccggactctagagccaccatggtgctgcagacccaggt gttc-3′ L-EKL-R:(SEQ ID NO: 119) 5′-caggtacaggttcttgttctcgttttccaggctctggctgcttctgcagc-3′ Primer set B L-EKL-F: (SEQ ID NO: 120)5′-gaaaacgagaacaagaacctgtacctgaactggtatctgcag aagcccg-3′ L inf-R:(SEQ ID NO: 121) 5′-tagcctcccccgtttaaacgggcccctaacactcccccctg-3′Primer set C L inf-F: (SEQ ID NO: 118)5′-gcctccggactctagagccaccatggtgctgca gacccaggtgttc-3′ L inf-R:(SEQ ID NO: 212) 5′-tagcctcccccgtttaaacgggcccctaacact cccccctg-3′

(Example 10) Preparation of Humanized MAb1 10-7-1) Production ofHumanized MAb1

FreeStyle 293-F cells (Invitrogen Corp.) were subcultured and culturedaccording to the manual.

1.2×10⁹ cells of FreeStyle 293-F cells (Invitrogen Corp.) at thelogarithmic growth phase were inoculated to 3 L Fernbach ErlenmeyerFlask (Corning Inc.), diluted with FreeStyle 293 expression medium(Invitrogen Corp.) into 1.0×10⁶ cells/mL, and then shake-cultured in an8% CO₂ incubator at 90 rpm at 37° C. for 1 hour. 3.6 mg ofpolyethyleneimine (Polysciences, Inc., #24765) was dissolved in 20 mL ofOpti-Pro SFM medium (Invitrogen Corp.). Next, an H chain expressionvector (0.4 mg) and an L chain expression vector (0.8 mg) prepared usingPureLink HiPure Plasmid kit (Invitrogen Corp.) were suspended in 20 mLof Opti-Pro SFM medium (Invitrogen Corp.). 20 mL of the expressionvector/Opti-Pro SFM mixture solution was added to 20 mL of thepolyethyleneimine/Opti-Pro SFM mixture solution, and the mixture wasgradually stirred, further left for 5 minutes, and then added to theFreeStyle 293-F cells. The cells were shake-cultured in an 8% CO₂incubator at 90 rpm at 37° C. for 7 days, and the obtained culturesupernatant was filtered through Disposable Capsule Filter (Advantec,#CCS-045-E1H), and purified in the same way as in 7)-2.

The humanized MAb1 antibody obtained by the combination betweenpCMA-G2/hMAb1-H1 and pCMA-LK/hMAb1-L1 was designated as “H-1040”. Thehumanized MAb1 antibody obtained by the combination betweenpCMA-G2/hMAb1-H2 and pCMA-LK/hMAb1-L1 was designated as “H-1140”. Thehumanized MAb1 antibody obtained by the combination betweenpCMA-G2/hMAb1-H2 and pCMA-LK/hMAb1-L2 was designated as “H-1143”. Thehumanized MAb1 antibody obtained by the combination betweenpCMA-G2/hMAb1-H3 and pCMA-LK/hMAb1-L1 was designated as “H-2040”. Thehumanized MAb1 antibody obtained by the combination betweenpCMA-G2/hMAb1-H4 and pCMA-LK/hMAb1-L1 was designated as “H-2140”. Thehumanized MAb1 antibody obtained by the combination betweenpCMA-G2/hMAb1-H4 and pCMA-LK/hMAb1-L2 was designated as “H-2143”.

(Example 11) Property of Anti-ROBO4 Humanized Antibody 11)-1 BindingAffinity

The dissociation constant between each antibody and rROBO4-ECD wasdetermined with the antibody immobilized as a ligand and the antigen asan analyte using Biacore 3000 (manufactured by GE HealthcareBio-Sciences Corp.). Approximately 80 RU of the antibody was bound viaan anti-human IgG antibody (manufactured by GE Healthcare Bio-SciencesCorp.) immobilized on a sensor chip CM5 (manufactured by GE HealthcareBio-Sciences Corp.) by an amine coupling method. The running buffer usedwas PBS containing 0.05% Surfactant P20. Dilution series solutions(0.1-200 nM) of the antigen were added at a flow rate of 30 μL/min. for300 seconds onto the antibody-bound chip. Subsequently, a dissociationphase was monitored for 300 seconds. 3 M MgCl₂ was added thereto as aregeneration solution at a flow rate of 10 μL/min. for 30 seconds. Datawas analyzed using the 1:1 binding model of analysis software(BIAevaluation Software, version 4.1) to calculate an association rateconstant kon, a dissociation rate constant koff, and a dissociationconstant (K_(D); K_(D)=koff/kon). As a result, the K_(D) value was 0.41nM for H-1040, 3.5 nM for H-1143, 3.9 nM for H-1140, 0.40 nM for H-2040,1.7 nM for H-2143, and 1.8 nM for H-2140.

11)-2 Activation of ROBO4 Downstream Signal

This test was conducted according to the method of 4)-1 except that:human IgG was used as a negative control; and Human IgG, H-1040, H-1140,H-1143, H-2040, H-2140, H-2143, or cMAb1-2 was added at a finalconcentration of 0.63 μg/mL to a medium. As a result, the negativecontrol human IgG did not influence the IL-8 promoter activity in thecells transiently expressing human ROBO4, whereas all the anti-ROBO4humanized antibodies increased the IL-8 promoter activity at a levelequivalent to cMAb1-2 (FIG. 49). These results demonstrated that all theanti-ROBO4 humanized antibodies activated the downstream signal ofROBO4, as in MAb1.

11)-3 HUVEC Migration Test

This test was conducted according to the method of 4)-2 except that:human IgG was used as a negative control; human IgG, H-1143, H-2140, orH-2143 was added at a final concentration of 0.5 μg/mL to a medium; andthe fluorescence intensity (excitation wavelength/fluorescentwavelength: 485 nm/538 nm) of each well was measured using a platereader (EnVision: Perkin Elmer, Inc.). As a result, H-1143, H-2140, orH-2143 suppressed the migration of HUVEC induced by bFGF (FIG. 50).Additionally, H-1140 showed the suppressive activity against HUVECmigration. These results demonstrated that H-1140, H-1143, H-2140, orH-2143 suppressed the migration of HUVEC.

11)-4 Cross-Species Reactivity

11)-4-1 Preparation of Antigen Gene-Expressing Cell

The cells were prepared according to the method of 4)-3-1.

11)-4-2 Flow Cytometry Analysis

This test was conducted according to the method of 4)-3-2 except that:human IgG was used as a negative control; FITC-AffiniPure GoatAnti-Human IgG, Fcγ Fragment Specific (manufactured by JacksonImmunoResearch Laboratories, Inc.) was used as a secondary antibody; anda flow cytometer (FC500; manufactured by Beckman Coulter, Inc.) was usedas a detector. As a result, H-1143, H-2140, or H-2143 did not bind tomouse ROBO4 or rat ROBO4, but was shown to bind to human ROBO4 andcynomolgus monkey ROBO4, as in the parent antibody MAb1 (FIGS. 51, 52,and 53). Additionally, H-1140 showed the same result as H-1143, H-2140and H-2143.

11)-5 Binding Specificity of H-1140, H-1143, H-2140, or H-2143

11)-5-1 Preparation of Antigen Gene-Expressing Cell

The cells were prepared according to the method of 4)-4-1 except thatpCI-FLAG-hROBO4-28 was used as a human ROBO4 expression vector.

11)-5-2 Flow Cytometry Analysis

This test was conducted according to the method of 4)-4-2 except that:human IgG or mouse IgG2A was used as a negative control;Fluorescein-conjugated goat IgG fraction to mouse IgG (manufactured byCappel Laboratories, Inc.) or FITC-AffiniPure Goat Anti-Human IgG, FcγFragment Specific was used as a secondary antibody; and a flow cytometer(FC500) was used as a detector. As a result, H-1143, H-2140, or H-2143did not bind to hROBO1, hROBO2, or hROBO3, but was shown to specificallybind to hROBO4, as in the parent antibody MAb1 (FIG. 54). Additionally,H-1140 showed the same result as H-1143, H-2140 and H-2143. In thiscontext, hROBO4, hROBO1, hROBO2, and hROBO3 were each confirmed to beexpressed on the cell membrane using the positive control antibody (FIG.54).

11)-6 Drug Efficacy Evaluation in Monkey Models with Laser-InducedChoroidal Neovascularization

11)-6-1 Anesthesia

Medetomidine hydrochloride was intramuscularly injected at a dose of0.08 mg/kg to each cynomolgus monkey. 15 minutes thereafter, ketaminehydrochloride was intramuscularly injected thereto at a dose of 15mg/kg.

11)-6-2 Model Preparation

Each cynomolgus monkey anesthetized in 11)-6-1 was retained on astainless operating table in the supine position. 5 mg/mL tropicamide-5mg/mL phenylephrine hydrochloride mixture eye drops were applied to theeyes for mydriasis. The macular region of the retina was thermallydamaged by laser irradiation (quantity of heat irradiated: 500 mW,irradiation time: 0.1 seconds, spot size: 50 μm, the number of spots: 9spots) using a green laser photocoagulator OcuLight GLx. After theoperation, Cravit eye drops were added dropwise to the operated eyes.

11)-6-3 Administration of Test Substance

At day 7 after the model preparation, each cynomolgus monkey wasanesthetized and then retained on a stainless operating table in thesupine position. Then, PA IODO Ophthalmic and Eye washing Solution(Nitten Pharmaceutical Co., Ltd.) diluted 4-fold with sterile purifiedwater was applied to the eyes to disinfect the external eyes. A 33Gneedle was inserted into the vitreous body from the conjunctiva, andsaline or 0.05 mL of H-2143 adjusted to 1.1 mg/0.05 mL was injectedthereto using a 1-mL syringe. After completion of the administration,Rinderon-A Ointment for Eye and Ear was applied to the conjunctiva usinga swab and spread throughout the surfaces of the eyes by the opening andclosing of the eyes.

11)-6-4 Drug Efficacy Evaluation

At days 7, 14, and 21 after the model preparation, the ocular fundus wasphotographed by a routine method using a hybrid fundus camera CX-1 underanesthesia. Then, fluorescein was intravenously injected thereto at adose of 0.05 mL/Kg. After completion of the intravenous injection offluorescein, fluorescent angiography was performed every 1 minute up to10 minutes later, and image data was stored. From the image data,severity was classified at a scale of grades 1 to 5 based on thefluorescence intensity of each laser-irradiated site at whichfluorescein accumulated according to the method of Zahn G et al. (Zahn Get al., Arch. Ophthalmol. 2009 127: 1329-1335). Then, the ratio of asite corresponding to grades 4 and 5 in the severity classificationamong all the laser-irradiated sites was calculated on the percentagebasis to evaluate choroidal neovascularization. As a result of comparingchoroidal neovascularization between the saline-administered group andthe H-2143-administered group, the ratio of grades 4 and 5 increased inthe saline-administered group with a lapse of time from the modelpreparation, whereas such increase was not observed in theH-2143-administered group. This means that the administration of H-2143suppressed laser-induced choroidal neovascularization (FIG. 55).

INDUSTRIAL APPLICABILITY

Use of an antibody provided by the present invention enables thetreatment or prevention of an angiogenic disease such as exudativeage-related macular degeneration, diabetic retinopathy, macular edema,benign or malignant tumor, atherosclerosis, retrolental fibroplasia,angioma, chronic inflammation, ocular neovascular disease, proliferativeretinopathy, neovascular glaucoma, immune rejection of a corneal tissuetransplant or other tissue transplants, rheumatoid arthritis, psoriasis,acute inflammation, sepsis, or obesity, and the examination or diagnosisof the angiogenic disease.

FREE TEXT OF SEQUENCE LISTING

SEQ ID NO: 1: Nucleotide sequence of full-length human ROBO4 cDNA (FIG.13).

SEQ ID NO: 2: Amino acid sequence of human ROBO4 (FIG. 14).

SEQ ID NO: 3: Nucleotide sequence encoding the amino acid sequence ofN-terminal FLAG-tagged full-length human ROBO4.

SEQ ID NO: 4: Amino acid sequence of N-terminal FLAG-tagged full-lengthhuman ROBO4.

SEQ ID NO: 5: Nucleotide sequence encoding the amino acid sequence of anN-terminal FLAG-tagged extracellular region/domain deletion variant ofhuman ROBO4 consisting of an amino acid sequence of amino acid Nos. 46to 1007 of SEQ ID NO: 2.

SEQ ID NO: 6: Amino acid sequence of an N-terminal FLAG-taggedextracellular region/domain deletion variant of human ROBO4 consistingof an amino acid sequence of amino acid Nos. 46 to 1007 of SEQ ID NO: 2.

SEQ ID NO: 7: Nucleotide sequence encoding the amino acid sequence of anN-terminal FLAG-tagged extracellular region/domain deletion variant ofhuman ROBO4 consisting of an amino acid sequence of amino acid Nos. 132to 1007 of SEQ ID NO: 2.

SEQ ID NO: 8: Amino acid sequence of an N-terminal FLAG-taggedextracellular region/domain deletion variant of human ROBO4 consistingof an amino acid sequence of amino acid Nos. 132 to 1007 of SEQ ID NO: 2

SEQ ID NO: 9: Nucleotide sequence encoding the amino acid sequence of anN-terminal FLAG-tagged extracellular region/domain deletion variant ofhuman ROBO4 consisting of an amino acid sequence of amino acid Nos. 210to 1007 of SEQ ID NO: 2.

SEQ ID NO: 10: Amino acid sequence of an N-terminal FLAG-taggedextracellular region/domain deletion variant of human ROBO4 consistingof an amino acid sequence of amino acid Nos. 210 to 1007 of SEQ ID NO:2.

SEQ ID NO: 11: Nucleotide sequence encoding the amino acid sequence ofan N-terminal FLAG-tagged extracellular region/domain deletion variantof human ROBO4 consisting of an amino acid sequence of amino acid Nos.225 to 1007 of SEQ ID NO: 2.

SEQ ID NO: 12: Amino acid sequence of an N-terminal FLAG-taggedextracellular region/domain deletion variant of human ROBO4 consistingof an amino acid sequence of amino acid Nos. 225 to 1007 of SEQ ID NO:2.

SEQ ID NO: 13: Nucleotide sequence encoding the amino acid sequence ofan N-terminal FLAG-tagged extracellular region/domain deletion variantof human ROBO4 consisting of an amino acid sequence of amino acid Nos.341 to 1007 of SEQ ID NO: 2.

SEQ ID NO: 14: Amino acid sequence of an N-terminal FLAG-taggedextracellular region/domain deletion variant of human ROBO4 consistingof an amino acid sequence of amino acid Nos. 341 to 1007 of SEQ ID NO:2.

SEQ ID NO: 15: Nucleotide sequence of mouse ROBO4 cDNA.

SEQ ID NO: 16: Amino acid sequence of mouse ROBO4.

SEQ ID NO: 17: Nucleotide sequence of rat ROBO4 cDNA.

SEQ ID NO: 18: Amino acid sequence of rat ROBO4.

SEQ ID NO: 19: Nucleotide sequence of monkey ROBO4 cDNA1.

SEQ ID NO: 20: Monkey ROBO4 amino acid sequence 1.

SEQ ID NO: 21: Nucleotide sequence of monkey ROBO4 cDNA2.

SEQ ID NO: 22: Monkey ROBO4 amino acid sequence 2.

SEQ ID NO: 23: Nucleotide sequence of human ROBO1 cDNA.

SEQ ID NO: 24: Amino acid sequence of human ROBO1.

SEQ ID NO: 25: Nucleotide sequence of human ROBO2 cDNA.

SEQ ID NO: 26: Amino acid sequence of human ROBO2.

SEQ ID NO: 27: Nucleotide sequence of human ROBO3 cDNA.

SEQ ID NO: 28: Amino acid sequence of human ROBO4.

SEQ ID NO: 29: Nucleotide sequence of an IL-8 promoter region.

SEQ ID NO: 30: Nucleotide sequence of cDNA encoding MAb1 heavy chainvariable region (FIG. 15).

SEQ ID NO: 31: Amino acid sequence of MAb1 heavy chain variable region(FIG. 16).

SEQ ID NO: 32: Nucleotide sequence of cDNA encoding MAb1 light chainvariable region (FIG. 17).

SEQ ID NO: 33: Amino acid sequence of MAb1 light chain variable region(FIG. 18).

SEQ ID NO: 34: Nucleotide sequence comprising cDNA encoding the aminoacids of human κ chain secretory signal and human κ chain constantregion.

SEQ ID NO: 35: Nucleotide sequence comprising cDNA encoding the aminoacids of human heavy chain signal sequence and human IgG1 constantregion.

SEQ ID NO: 36: Nucleotide sequence comprising cDNA encoding the aminoacids of human heavy chain signal sequence and human IgG2 constantregion.

SEQ ID NO: 37: Nucleotide sequence of cDNA encoding cMAb1 light chain(FIG. 19).

SEQ ID NO: 38: Amino acid sequence of cMAb1 light chain (FIG. 20).

SEQ ID NO: 39: Nucleotide sequence of cDNA encoding cMAb1-1 heavy chain(FIG. 21).

SEQ ID NO: 40: Amino acid sequence of cMAb1-1 heavy chain (FIG. 22).

SEQ ID NO: 41: Nucleotide sequence of cDNA encoding cMAb1-2 heavy chain(FIG. 23).

SEQ ID NO: 42: Amino acid sequence of cMAb1-2 heavy chain (FIG. 24).

SEQ ID NO: 43: Nucleotide sequence encoding the amino acid sequence ofMAb1 heavy chain CDRH1.

SEQ ID NO: 44: Amino acid sequence of MAb1 heavy chain CDRH1 (FIG. 25).

SEQ ID NO: 45: Nucleotide sequence encoding the amino acid sequence ofMAb1 heavy chain CDRH2.

SEQ ID NO: 46: Amino acid sequence of MAb1 heavy chain CDRH2 (FIG. 26).

SEQ ID NO: 47: Nucleotide sequence encoding the amino acid sequence ofMAb1 heavy chain CDRH3.

SEQ ID NO: 48: Amino acid sequence of MAb1 heavy chain CDRH3 (FIG. 27).

SEQ ID NO: 49: Nucleotide sequence encoding the amino acid sequence ofMAb1 light chain CDRL1.

SEQ ID NO: 50: Amino acid sequence of MAb1 light chain CDRL1 (FIG. 28).

SEQ ID NO: 51: Nucleotide sequence encoding the amino acid sequence ofMAb1 light chain CDRL2.

SEQ ID NO: 52: Amino acid sequence of MAb1 light chain CDRL2 (FIG. 29).

SEQ ID NO: 53: Nucleotide sequence encoding the amino acid sequence ofMAb1 light chain CDRL3.

SEQ ID NO: 54: Amino acid sequence of MAb1 light chain CDRL3 (FIG. 30).

SEQ ID NO: 55: Nucleotide sequence of cDNA encoding hMAb1-H1-type heavychain (FIG. 31).

SEQ ID NO: 56: Amino acid sequence of hMAb1-H1-type heavy chain (FIG.32).

SEQ ID NO: 57: Nucleotide sequence of cDNA encoding hMAb1-H2-type heavychain (FIG. 33).

SEQ ID NO: 58: Amino acid sequence of hMAb1-H2-type heavy chain (FIG.34).

SEQ ID NO: 59: Nucleotide sequence of cDNA encoding hMAb1-H3-type heavychain (FIG. 35).

SEQ ID NO: 60: Amino acid sequence of hMAb1-H3-type heavy chain (FIG.36).

SEQ ID NO: 61: Nucleotide sequence of cDNA encoding hMAb1-H4-type heavychain (FIG. 37).

SEQ ID NO: 62: Amino acid sequence of hMAb1-H4-type heavy chain (FIG.38).

SEQ ID NO: 63: Nucleotide sequence of cDNA encoding hMAb1-L1-type lightchain (FIG. 39).

SEQ ID NO: 64: Amino acid sequence of hMAb1-L1-type heavy chain (FIG.40).

SEQ ID NO: 65: Nucleotide sequence of cDNA encoding hMAb1-L2-type heavychain (FIG. 41).

SEQ ID NO: 66: Amino acid sequence of hMAb1-L2-type heavy chain (FIG.42).

SEQ ID NO: 67: CDRH1 of hMAb1-H2 or hMAb1-H4-type heavy chain (FIG. 43).

SEQ ID NO: 68: CDRH2 of hMAb1-H2 or hMAb1-H4-type heavy chain (FIG. 44).

SEQ ID NO: 69: CDRH3 of hMAb1-H2 or hMAb1-H4-type heavy chain (FIG. 45).

SEQ ID NO: 70: CDRL1 of hMAb1-L2-type light chain (FIG. 46).

SEQ ID NO: 71: CDRL2 of hMAb1-L2-type light chain (FIG. 47).

SEQ ID NO: 72: CDRL3 of hMAb1-L2-type light chain (FIG. 48).

The invention claimed is:
 1. A monoclonal antibody or an antigen bindingfragment thereof that specifically binds to human ROBO4 and comprises aheavy chain and a light chain, wherein: a) said heavy chain comprisesSEQ ID NO: 44 (CDRH1), SEQ ID NO: 46 (CDRH2), and SEQ ID NO: 48 (CDRH3)and said light chain comprises SEQ ID NO: 50 (CDRL1), SEQ ID NO: 52(CDRL2), and SEQ ID NO: 54 (CDRL3); b) said heavy chain comprises SEQ IDNO: 44 (CDRH1), SEQ ID NO: 68 (CDRH2), and SEQ ID NO: 48 (CDRH3) andsaid light chain comprises SEQ ID NO: 50 (CDRL1), SEQ ID NO: 52 (CDRL2),and SEQ ID NO: 54 (CDRL3); c) said heavy chain comprises SEQ ID NO: 44(CDRH1), SEQ ID NO: 68 (CDRH2), and SEQ ID NO: 48 (CDRH3) and said lightchain comprises SEQ ID NO: 70 (CDRL1), SEQ ID NO: 52 (CDRL2), and SEQ IDNO: 54 (CDRL3); or d) said heavy chain comprises SEQ ID NO: 44 (CDRH1),SEQ ID NO: 46 (CDRH2), and SEQ ID NO: 48 (CDRH3) and said light chaincomprises SEQ ID NO: 70 (CDRL1), SEQ ID NO: 52 (CDRL2), and SEQ ID NO:54 (CDRL3).
 2. The monoclonal antibody or the antigen binding fragmentthereof according to claim 1, wherein the ROBO4 protein is the humanROBO4 protein.
 3. The monoclonal antibody or the antigen bindingfragment thereof according to claim 1, wherein the ROBO4 protein is aprotein consisting of: a) amino acid Nos. 1 to 1007 of SEQ ID NO: 2; orb) amino acid Nos. 46 to 1007 of SEQ ID NO:
 2. 4. The monoclonalantibody or the antigen binding fragment thereof according to claim 3,wherein the antibody or the antigen binding fragment thereof binds to asite consisting of amino acid Nos. 132 to 209 of SEQ ID NO:
 2. 5. Themonoclonal antibody or the antigen binding fragment thereof according toclaim 1, wherein said antibody comprises a heavy chain comprising one ormore modifications selected from the group consisting of N-linkedglycosylation, O-linked glycosylation, N-terminal processing, C-terminalprocessing, deamidation, isomerization of aspartic acid, oxidation ofmethionine, and amidation of a proline residue.
 6. The monoclonalantibody or the antigen binding fragment thereof according to claim 1,wherein the antibody comprises a heavy chain variable region comprisingSEQ ID NO: 31, and a light chain variable region comprising SEQ ID NO:33.
 7. The monoclonal antibody or the antigen binding fragment thereofaccording to claim 1, wherein the antibody or the antigen bindingfragment thereof is: a) a chimeric antibody or an antigen bindingfragment thereof; b) a humanized antibody or an antigen binding fragmentthereof, or c) a human antibody or an antigen binding fragment thereof.8. The monoclonal antibody or the antigen binding fragment thereofaccording to claim 1, wherein the antibody comprises a human IgG1 orhuman IgG2 heavy chain constant region.
 9. The antibody according toclaim 1, wherein said antibody lacks one to several tencarboxyl-terminal amino acid(s) of said heavy chain.
 10. Apharmaceutical composition comprising a monoclonal antibody or anantigen binding fragment thereof according to claim 1 and apharmaceutical material.
 11. The pharmaceutical composition according toclaim 10, wherein said composition comprises a further therapeuticagent.
 12. The monoclonal antibody or antigen binding fragment accordingto claim 1, wherein said heavy chain comprises SEQ ID NO: 44 (CDRH1),SEQ ID NO: 46 (CDRH2), and SEQ ID NO: 48 (CDRH3) and said light chaincomprises SEQ ID NO: 50 (CDRL1), SEQ ID NO: 52 (CDRL2), and SEQ ID NO:54 (CDRL3).
 13. The monoclonal antibody or antigen binding fragmentaccording to claim 1, wherein said heavy chain comprises SEQ ID NO: 44(CDRH1), SEQ ID NO: 68 (CDRH2), and SEQ ID NO: 48 (CDRH3) and said lightchain comprises SEQ ID NO: 50 (CDRL1), SEQ ID NO: 52 (CDRL2), and SEQ IDNO: 54 (CDRL3).
 14. The monoclonal antibody or antigen binding fragmentaccording to claim 1, wherein said heavy chain comprises SEQ ID NO: 44(CDRH1), SEQ ID NO: 68 (CDRH2), and SEQ ID NO: 48 (CDRH3) and said lightchain comprises SEQ ID NO: 70 (CDRL1), SEQ ID NO: 52 (CDRL2), and SEQ IDNO: 54 (CDRL3).
 15. The monoclonal antibody or antigen binding fragmentaccording to claim 1, wherein said heavy chain comprises SEQ ID NO: 44(CDRH1), SEQ ID NO: 46 (CDRH2), and SEQ ID NO: 48 (CDRH3) and said lightchain comprises SEQ ID NO: 70 (CDRL1), SEQ ID NO: 52 (CDRL2), and SEQ IDNO: 54 (CDRL3).
 16. A method for treating an angiogenic diseasecomprising administering to a subject in need thereof an effectiveamount of an antibody or an antigen binding fragment thereof accordingto claim 1 or administering a composition comprising a pharmaceuticalmaterial and said antibody to said subject.
 17. The method according toclaim 16, wherein said angiogenic disease is exudative age-relatedmacular degeneration, diabetic retinopathy, macular edema, benign ormalignant tumor, atherosclerosis, retrolental fibroplasia, angioma,chronic inflammation, ocular neovascular disease, proliferativeretinopathy, neovascular glaucoma, immune rejection of a corneal tissuetransplant or other tissue transplants, rheumatoid arthritis, psoriasis,acute inflammation, sepsis, or obesity.
 18. The method according toclaim 16, wherein said angiogenic disease is exudative age-relatedmacular degeneration, diabetic retinopathy, macular edema, retrolentalfibroplasia, ocular neovascular disease, proliferative retinopathy,neovascular glaucoma, or immune rejection of a corneal tissuetransplant.
 19. The method according to claim 16, wherein said methodcomprising administering the subject a further therapeutic agent.